Characterization of 4-demethylwyosine Synthase, a Radical S-adenosyl-l-methionine Enzyme Involved in the Modification of tRNA
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Authors Young, Anthony Peter
Publisher The University of Arizona.
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CHARACTERIZATION OF 4-DEMETHYLWYOSINE SYNTHASE, A
RADICAL S-ADENOSYL-L-METHIONINE ENZYME INVOLVED IN THE
MODIFICATION OF tRNA
by
Anthony P. Young
______
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN BIOCHEMISTRY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2016
1 THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Anthony P. Young, titled Characterization of 4- demethylwyosine synthase, a radical S-adenosyl-L-methionine enzyme involved in the modification of tRNA and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.
______Date: 7/11/2016 John Jewett
______Date: 7/11/2016 Vahe Bandarian
______Date: 7/11/2016 Matthew Cordes
______Date: 7/11/2016 Megan McEvoy
______Date: 7/11/2016 William Montfort
Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
______Date: 7/11/2016 Dissertation Director: John Jewett
2 STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.
SIGNED: Anthony P. Young
3 TABLE OF CONTENTS
LIST OF TABLES ...... 9
LIST OF FIGURES ...... 10
ABSTRACT ...... 13
CHAPTER 1: INTRODUCTION TO WYOSINE BASES ...... 15
1.1 Modified RNA bases ...... 16
1.2 Discovery of wyosine derivatives ...... 19
1.3 Biosythesis of wyosine derivatives ...... 20
1.3.a N-methylguanosine (m1G) ...... 20
1.3.b 4-demethylwyosine (imG-14) ...... 22
1.4 Wyosine biosynthesis in eukaryotes ...... 26
1.4.a Wybutosine biosynthesis (yW) ...... 26
1.4.a.i 7-aminocarboxypropyldemethylwyosine (yW-86) ...... 28
1.4.a.ii 7-aminocarboxypropylwyosine (yW-72) ...... 29
1.4.a.iii Wybutosine (yW) - yeast ...... 30
1.4.a.iv Wybutosine (yW) - plants ...... 31
1.4.b Hydroxywybutosine (OHyW) ...... 32
1.4.c Wyosine (imG) ...... 34
1.5 Wyosine biosynthesis in archaea ...... 34
4 1.5.a 4-demethylwyosine (imG-14) ...... 36
1.5.b Wyosine (imG) ...... 36
1.5.c 7-methylwyosine (mimG) ...... 37
1.5.d Iso-wyosine (imG2) ...... 37
1.6 Wyosine derivatives in mitrochondria ...... 38
1.7 Biological role of wyosine derivatives ...... 39
CHAPTER 2: RADICAL S-ADENOSYL-L-METHIONINE ENZYMES .... 41
2.1 Common features of radical SAM enzymes ...... 42
2.2 Classes of radical SAM enzymes ...... 44
2.3 Radical SAM enzymes with auxiliary clusters ...... 46
2.3.a Sulfur insertion ...... 46
2.3.a.i BioB (biotin synthase) ...... 47
2.3.a.ii LipA (lipoyl synthase) ...... 49
2.3.a.iii Methylthiotransferases (MiaB and RimO) ...... 49
2.3.b Coordination of substrate ...... 50
2.3.b.i MoaA (molybdopterin cofactor biosynthesis) ...... 50
2.3.c Peptide and protein modifying radical SAM enzymes ...... 53
2.3.c.i AlbA (subtilosin A biosynthesis) ...... 53
2.3.c.ii SkfB (sporulation killing factor biosynthesis) ...... 55
5 2.3.c.iii ThnB (thurincin H maturation) ...... 56
2.3.c.iv Tte1186 (SCIFF peptide modification) ...... 57
2.3.c.v PqqE (pyrroloquinoline quinone cofactor biosynthesis) ... 58
2.3.c.vi StrB (streptide biosynthesis) ...... 59
2.3.c.vii MftC (mycofactocin biosynthesis) ...... 60
2.3.d Radical dehydrogenase subfamily ...... 60
2.3.d.i AtsB (anaerobic sulfatase maturation enzyme) ...... 61
2.3.d.ii anSMEcpe (cys anaerobic sulfatase maturation enzyme) 62
2.3.d.iii BtrN (2-deoxy-scyllo-inosamine biosynthesis) ...... 63
2.4 Conclusion ...... 64
CHAPTER 3: PYRUVATE IS THE SOURCE OF THE TWO CARBONS
THAT ARE REQUIRED FOR CONVERSION OF N-
METHYLGUANOSINE TO THE IMIDAZOLINE TRICYCLIC BASE OF
4-DEMETHYLWYOSINE ...... 65
3.1 Abstract ...... 66
3.2 Introduction ...... 66
3.3 Results and discussion ...... 68
3.4 Materials and methods ...... 78
6 CHAPTER 4: MECHANISTIC STUDIES OF THE RADICAL SAM
ENZYME 4-DEMETHYLWYOSINE SYNTHASE REVEALS THE SITE OF
HYDROGEN ATOM ABSTRACTION ...... 86
4.1 Abstract ...... 87
4.2 Introduction ...... 87
4.3 Results and discussion ...... 90
4.4 Materials and methods ...... 103
CHAPTER 5: BIOCHEMICAL CHARACTERIZATION OF A SCHIFF
BASE INTERMEDIATE THAT FORMS DURING 4-
DEMETHYLWYSOINE BIOSYNTHESIS ...... 111
5.1 Abstract ...... 112
5.2 Introduction ...... 112
5.3 Results and discussion ...... 119
5.4 Materials and methods ...... 131
CHAPTER 6: MUTAGENESIS STUDY OF THE CYSTEINE RESIDUES
HYPOTHESIZED TO LIGATE THE TWO IRON SULFUR CLUSTERS IN
TYW1 ...... 138
6.1 Abstract ...... 139
6.2 Introduction ...... 139
6.3 Results ...... 144
7 6.4 Discussion ...... 150
6.5 Materials and methods ...... 154
CHAPTER 7: CONCLUSIONS AND FUTURE WORK ...... 158
7.1 Conclusions ...... 159
7.2 Future directions ...... 162
7.2.a Archaeal homologs of TYW1 ...... 162
7.2.b Eukaryal homologs of TYW1 ...... 163
APPENDIX A ...... 165
REFERENCES ...... 172
8 LIST OF TABLES
Table 5.1: Primers used to make C195S and K41A variants ...... 133
Table 6.1: Iron and sulfide analysis of cysteine mutants ...... 149
Table 6.2: Primers used to make cysteine mutants ...... 156
9 LIST OF FIGURES
Figure 1.1: Examples of modified RNA bases ...... 17
Figure 1.2: RNA modifications in tRNA ...... 18
Figure 1.3: Wyosine derivatives ...... 21
Figure 1.4: Sequence alignment of TYW1 ...... 24
Figure 1.5: Biosynthesis of wyosine derivatives in eukaryotes ...... 27
Figure 1.6: Biosynthesis of wyosine derivatives in archaea ...... 35
Figure 2.1: Examples of reactions catalyzed by radical SAM enzymes ...... 43
Figure 2.2: Catalytic cycle of radical SAM enzymes ...... 45
Figure 2.3: Sulfur insertion catalyzed by radical SAM enzymes ...... 48
Figure 2.4: Conversion of GTP to precursor Z ...... 51
Figure 3.1: Biosynthetic pathway of yW ...... 67
Figure 3.2: UV-visible spectrum of TYW1 ...... 70
Figure 3.3: Extracted ion chromatograms showing results of TYW1 incubation with proposed substrates ...... 71
Figure 3.4: Extracted ion chromatograms showing imG-14 production is dependent on TYW1 ...... 72
Figure 3.5: Extracted ion chromatograms showing incorporation of C2 and C3 into imG-14 ...... 74
Figure 3.6: Putative mechanism for transformation of m1G to imG-14 ...... 76
10 Figure 3.7: Codon optimized DNA and protein sequence of TRM5 ...... 79
Figure 3.8: Codon optimized DNA and protein sequence of TYW1 ...... 80
Figure 4.1: Biosynthesis of imG-14 ...... 88
Figure 4.2: dAdoH retention time ...... 92
Figure 4.3: Mass spectrum of dAdoH ...... 93
Figure 4.4: Mass spectrum of imG-14 ...... 95
Figure 4.5: Extracted ion chromatograms of m1G ...... 96
Figure 4.6: Mass spectrum of m1G ...... 97
Figure 4.7: Mass spectrum of imG-14 when digested in D2O ...... 99
Figure 4.8: Extracted ion chromatograms showing retention of deuterium in imG-14 from pyruvate ...... 101
Figure 4.9: Proposed mechanism for formation of imG-14 ...... 102
Figure 4.10: Mass spectrum of S-adenosyl-[methyl-D3]-methionine ...... 107
Figure 5.1: Biosynthetic pathway for yW from G ...... 114
Figure 5.2: Active site of M. jannaschii TYW1 ...... 117
Figure 5.3: Proposed mechanisms for conversion of m1G to imG-14 ...... 118
Figure 5.4: Extracted ion chromatogram at m/z 322 with C195S variant ... 120
Figure 5.5: Extracted ion chromatogram at m/z 322 with K41A variant .... 121
Figure 5.6: Possible peptide structures resulting from trypsin digest ...... 123
11 Figure 5.7: Total ion count and extracted ion chromatogram of trypsin digested
TYW1 ...... 125
Figure 5.8: Mass spectra of trypsin digested TYW1 ...... 126
Figure 5.9: Schiff base trapping controls ...... 129
Figure 6.1: TYW1 active site showing conserved cysteine and lysine ...... 142
Figure 6.2: Extracted ion chromatogram showing the cysteine mutant activity
...... 145
Figure 6.3: Extracted ion chromatogram showing cysteine mutant deoxyadenosine production ...... 147
Figure 6.4: TYW1 auxiliary cluster binding site ...... 152
12 ABSTRACT
Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N- methylguanosine to 4-demethylwyosine by the radical S-adenosyl-L-methionine enzyme TYW1.
When these studies were initiated, the substrate of TYW1 was unknown.
Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, 13C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct.
Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5’-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group.
13 The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate.
The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro.
It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of
N-methylguanosine to 4-demethylwyosine.
14 Chapter 1:
Introduction to wyosine RNA bases
15 1.1 Modified RNA bases
In addition to the four standard ribonucleic acid (RNA) bases there are over
150 documented modified RNA bases that are found throughout the three kingdoms of life1,2. Modified RNA bases range greatly in complexity from simple methylations of heteroatoms, requiring a single enzyme, to highly complex modifications that require multiple enzymes1,2. The modified bases play different roles depending upon where they are found. For example, methylation of C-8 of
A2503 in bacterial 23S ribosomal RNA (rRNA) provides antibiotic resistance3, while the modified base lysidine, a modified cytidine found in transfer ribonucleic acid (tRNA) encoding for isoleucine, changes its base pairing ability from guanosine to adenosine allowing for better translational fidelity4. These two modifications, along with other examples of both simple and complex modifications are shown in Figure 1.1. There are also many modified RNA bases whose roles are unknown.
The majority of modified RNA bases, over 100, are found in tRNA1,2. Many modified tRNA bases are conserved throughout all domains of life, both in structure and their location in tRNA. The bases play different roles depending upon where they are found in tRNA. Figure 1.2 shows the standard tRNA cloverleaf structure, with the circles representing different levels of modification in all of the tRNAs sequenced so far. The white circles represent positions in which a modified base has not been found, while red circles represent positions in which a modified base has been found, and black circles represent hypermodified positions, which means
16
Figure 1.1 Examples of modified RNA bases found in tRNA and rRNA.
17
Figure 1.2 The cloverleaf structure of tRNA with the white circles representing positions where no modified base has been found in all sequenced tRNAs, red circles representing positions where a modified base has been found, and black circles positions where more than 25% of the bases are modified.
18 that more than 25% of the bases found at that position are modified5. This shows that modifications are highly conserved in some regions of the cloverleaf, indicating that those modifications may play an important role. Those found in the anticodon loop region of the tRNA cloverleaf play roles related to decoding. For example, a large hypermodified base is found at position 37 of tRNAPhe in all domains of life. In bacteria a highly modified adenosine base is found at this position such as N6-threonylcarbamoyladenosine (see Figure 1.1 for structure), while in eukaryotes and archaea wyosine derivatives are found at this position6.
1.2 Discovery of wyosine derivatives
Wyosine derivatives are characterized by an aromatic tricyclic ring structure7. The first wyosine derivative to be discovered was wybutosine (yW) in tRNAPhe from baker’s yeast (S. cerevisiae) in 1967 during sequencing of phenylalanine encoding tRNA8-10. Subsequently three other wyosine derivatives; peroxywybutosine (o2yW), hydroxywybutosine (OHyW), and wyosine (imG);
7,11,12 were discovered in other eukaryotic phenylalanine encoding tRNA . The o2yW was originally found in rat liver in 197311, however in 1979 a different group analyzed rat liver tRNAPhe and found OHyW only, leading them to hypothesize that the o2yW is a degradation product of the OHyW and that o2yW is not a naturally occurring RNA base12.
In 1987 7-methylwyosine (mimG) was discovered in S. solfataricus, an archaeal strain13. Since their initial discovery in 1967 wyosine derivatives have been found in all sequenced archaea and eukaryotic tRNAPhe with the exception of
19 B. mori14,15 and D. melanogaster16, which both contain N-methylguanosine (m1G) in its place.
Figure 1.3.A-C shows all of the known wyosine derivatives, with Figure
1.3.A containing those only found in eukaryotes, Figure 1.3.B containing those found in both eukaryotes and archaea, and Figure 1.3.C those only found in archaea.
Those found in both eukaryotes are usually present as biosynthetic intermediates with the exceptions of yW, OHyW, and imG7,8,11,17.
1.3 Biosynthesis of wyosine derivatives
In 1973 it was discovered that wyosine base derivatives were derived from guanosine. Utilizing a mutant strain of S. cerevisiae deficient in GMP synthetase grown on [8-14C]-guanine the authors showed that both the guanosine bases and yW purified from this strain have similar levels of radioactivity, showing that yW, and probably all other wyosine derivatives, are derived from a guanosine residue18.
In both eukaryotes and archaea, the first two steps of wyosine biosynthesis are conserved. The first step is the methylation of guanosine 37 producing m1G by
TRM5 and the second step is the formation of 4-demethylwyosine (imG-14) by the radical S-adenosyl-L-methionine (SAM) enzyme TYW1.
1.3.a N-methylguanosine (m1G)
The first step in the biosynthesis of all wyosine derivatives is methylation of guanosine. In 1987 m1G containing tRNAPhe was identified as an intermediate in the biosynthesis of yW. Utilizing tRNA that was produced semi-synthetically,
20
Figure 1.3 The documented wyosine derivatives. Figure 1.3A shows the derivatives found only in eukaryotes, Figure 1.3B shows those found in both eukaryotes and archaea, and Figure 1.3C shows the wyosine derivatives found only in archaea.
21 containing no modifications in the anti-codon loop, and yeast lysate, tRNA containing m1G was successfully produced. The m1G containing tRNA could then be introduced into X. laevis oocytes, where the m1G would be converted to yW showing that m1G is a biosynthetic intermediate19. The gene responsible for m1G synthase in S. cerevisiae was identified in 2001 and subsequently homologs were found throughout all domains of life, as m1G is one of the modifications that is conserved throughout all domains of life20.
The second step in the biosynthesis of wyosine derivatives is catalyzed by the radical SAM enzyme 4-demethylwyosine synthase (TYW1). This step is common to all wyosine derivatives in both archaea and eukaryotes.
1.3.b 4-demethylwyosine (imG-14)
The enzyme responsible for the transformation of m1G into imG-14 and was discovered first in 200521. Bioinformatics, along with the knowledge that the modification, and subsequently the gene, was present in eukaryotic species such as
H. sapiens, S. cerevisiae, S. pombe, A. thaliana, and the archaeal species M. jannaschii, while absent in bacteria and D. melanogaster was used to identify the
S. cerevisiae gene YPL207w as a candidate for a gene involved in yW biosynthesis.
Analysis of bulk tRNA extracted from a DYPL207w S. cerevisiae strain showed no yW, while there was an accumulation of m1G. Subsequent in vivo complementation assays with the YPL207w gene present in trans on a plasmid restored yW production showing that YPL207w is a gene involved in yW biosynthesis21.
22 In 2006 a reverse genetic approach was employed to identify the genes responsible for the biosynthesis of yW. In this experiment S. cerevisiae genes of unknown function with homologs present in S. pombe were identified, 351 in total, and the S. cerevisiae deletion strains of each of these genes were screened for the presence of yW. This approach confirmed the role of YPL207w, which was renamed as TYW1, as a gene involved in the biosynthesis of yW17.
A sequence alignment of TYW1, shown in Figure 1.4, revealed a
CxxxCxxC motif, shown in blue, that is characteristic of the growing radical SAM superfamily22. The following is a brief description of radical SAM enzymes, with a greater description of the radical SAM superfamily following in Chapter 2.
Briefly, a CxxxCxxC motif coordinates a 4Fe-4S cluster that facilitates the reductive cleavage of SAM forming the highly reactive intermediate, 5′- deoxyadenosyl radical (dAdo•), and methionine. Hydrogen atom abstraction by dAdo• forms a substrate radical, which than goes on to form the final product23.
Analysis of TYW1 by PSI-BLAST shows that that the eukaryotic homologs of TYW1 contain two domains, an N terminal flavodoxin like domain and a C terminal radical SAM domain, while the archaeal homologs only have the radical
SAM domain17. As will be discussed in greater detail in Chapter 2, radical SAM enzymes require a reductant in order to be active. The N terminal domain of eukaryotic TYW1 is thought to act as the source of reductant, but this has yet to be experimentally confirmed.
Two crystal structures of TYW1 homologs from the archaeal species P. horikoshii24 and M. jannaschii25 have been solved. Neither of the structures
23 Mj 1 MI------Ph 1 MTI------Hs 1 MDPSADTWDLFSPLISLWINRFYIYLGFAVSISLWICVQIVIKT------Q--- Sc 1 MDG----FRVAGAIVVGALTAAYLYFGGRFSIALVIIVGYGIYCNEVSGGSQDSQEKLDLNKQQKKPCCSDKKIADGGKK *
Mj 3 ------Ph 4 ------KPG------Hs 46 ------G------KNLQE------Sc 77 TGGCCSDKKNGGGKGGGCCSSKGGKKGGCCSSKGGKKGGCCSSKKNIGDNENTATEVEKAVNYPVTVDFTEVFKKPTKKR
Mj 3 ------Ph 7 ------KITVQAN------Hs 52 K------SV-PKAAQDLMTNGYVSLQEKDIFVSGVKIFYGSQTGTAKGFATVLAEAVTSLDL---P Sc 157 SSTPKVFSKNSSSNSRVGKKLSVSKKIGPDGLIKSALTISNETLLSSQIYVLYSSLQGAASKAAKSVYDKLKELDELTNE
Mj 3 ------P------Ph 14 ------PNMP------Hs 107 VAIINLKEY-DPDDHLIEEVTSKNVCVFLVATYTDGLPTESAEWFCKWLEEASIDFRFGKTYLKG-MRYAVFGLGNS-AY Sc 294 PKLLNLDDLSDFDDYFINVPVENALYVLVLPSYDIDCP---LDYFLQTLEENANDFRVDSFPLRKLVGYTVLGLGDSESW *
Mj 4 ------Ph 18 ------Hs 184 ASHFNKVGKNVDKWLWMLGAHRVMSRGEGDCDVVKSKHGSIEADFRAWKTKFISQLQALQKGERKKSCGGHCKKGKCESH Sc 365 PEKFCYQAKRADHWISRLGGRRIFPLGKVC----MKTGGSA--KIDEWTSLLAETLK------
Mj 4 ------Ph 18 ------Hs 264 QHGSEEREEGSHEQDELHHRDTEEEEPFESSSEEEFGGEDHQSLNSIVDVEDLGKIMDHVKKEKREKEQQEEKSGLFRNM Sc 428 -----DDEPIIYEYDENA--DSEEDEEEG------NGSDELGDVEDIGGKGSNGKF------S
Mj 4 ------EEIYKILRKQRYQIDG-HTAVKLCGWVRKKMLEDKNCYKSKFYGIETHRCIQCTPSVIWCQQNCIFC Ph 18 ------KEVAELFRKQHYEIVGRHSGVKLCHWLKKSLTEGRFCYKQKFYGIHSHRCLQMTPVLAWCTHNCIFC Hs 344 GRNEDGERRAMITPALREALTKQGYQLIGSHSGVKLCRWTKSMLRGRGGCYKHTFYGIESHRCMETTPSLA-CANKCVFC Sc 523 GADEIKQMVAKD-SPTYKNLTKQGYKVIGSHSGVKICRWTKNELRGKGSCYKKSLFNIASSRCMELTPSLA-CSSKCVFC ** * * * ** * * *** * ** * * *
Mj 70 WRVLPRDIGIDISQIKEPKWEEPEVVYEKILAMHKRIIMGYAGVLDRVGEKKFKEALEPKHVAISLSGEPTLYPYLDELI Ph 85 WRPMENFLGTELP----QPWDDPAFIVEESIKAQRKLLIGYKGNP-KVDKKKFEEAWNPTHAAISLSGEPMLYPYMGDLV Hs 423 WRHHTNPVGTEWR----WKMDQPEMILKEAIENHQNMIKQFKGVP-GVKAERFEEGMTVKHCALSLVGEPIMYPEINRFL Sc 618 WRHGTNPVSKNWR----WEVDEPEYILENALKGHYSMIKQMRGVP-GVIAERFAKAFEVRHCALSLVGEPILYPHINKFI ** * * * * * ** *** **
Mj 150 KIFHKNGFTTFVVSNGILTDVIEKIE-----PTQLYISLDAYDLDSYRRICG-GKKEYWESILNTLDILKE---KKRTCI Ph 160 EEFHKRGFTTFIVTNGTIPERLEEMIKEDKLPTQLYVSITAPDIETYNSVNIPMIPDGWERILRFLELMRDL--PTRTVV Hs 498 KLLHQCKISSFLVTNAQFPAEIRNLE----PVTQLYVSVDASTKDSLKKIDRPLFKDFWQRFLDSLKALAVK--QQRTVY Sc 718 QLLHQKGITSFLVCNAQHPEALRNIV----KVTQLYVSIDAPTKTELKKVDRPLYKDFWERMVECLEILKTVQNHQRTVF * * * * *** * * * Mj 222 RTTLIRGYND-DILKFVELYERADVHFIELKSYMHVGYSQ---KRLKKEDMLQHDEILKLAKML-DEN----SSYKLIDD Ph 238 RLTLVKGENMHSPEKYAKLILKARPMFVEAKAYMFVGYSR---NRLTINNMPSHQDIREFAEALVKHL----PGYHIEDE Hs 572 RLTLVKAWNVDELQAYAQLVSLGNPDFIEVKGVTYCGESS--ASSLTMAHVPWHEEVVQFVHELVDLI----PEYEIACE Sc RLTLVKGFNMGDISAYADLVQRGLPSFIEVKGATFSGSSDGNGNPLTMQNIPFYEECVNFVKAFTTELQRRGLHYDLAAE * ** * * * * * * *
Mj 293 SEDSRVALLQNENRKINPKL------Ph 311 YEPSRVVLIMRDDVDPQGTGVEGRFIKH------Hs 646 HEHSNCLLIAHRKFKIVVNGGHGSIITA------SRSSSRNMKIVVD Sc HAHSNCLLIADTKFKINGE—-WHTHIDFDKFFVLLNSGKDFTYMDYLEKTPEWALFGNGGFAPGNTRVYRKDKKKQNKEN •
Mj ------Ph ------Hs QKRSAQRIIWPELLTGHYLVPVKEALIPRTQDIRERTNQRLFLDVEII Sc QETTTRETPLPPIPA------
Figure 1.4 Sequence alignment of TYW1 from M. jannaschii (Mj), P. horikoshii (Ph), H. sapian (Hs), and S. cerevisiae (Sc). The radical SAM cysteine motif is shown in blue and the cysteine motif hypothesized to coordinate the second cluster is shown in red.
24 contained electron density for the 4Fe-4S cluster that is required to form dAdo•.
The structures did however identify another cluster of three cysteines, shown in red in Figure 1.4, that were close enough to form a second iron sulfur cluster24,25. One group hypothesized that the second cluster was a 2Fe-2S cluster24 and the other a
4Fe-4S cluster25. A mutagenesis study, using in vivo complementation in a DTYW1
S. cerevisiae strain to detect activity through the presence of yW, confirmed that the six cysteine residues thought to coordinate the two clusters produce inactive protein when mutated to alanine residues25. A subsequent electron paramagnetic resonance study confirmed the presence of two 4Fe-4S clusters26 as predicted by
Suzuki et al25.
As the TYW1 gene was confirmed as 4-demethylwyosine synthase on the basis of in vivo complementation assays by two groups, the identity of the second substrate was not required for these assays to work and was not known at the time.
The transformation of m1G to imG-14 requires the addition of two carbons to form the imidazoline ring. The second substrate of TYW1 was identified as pyruvate and that study is the basis of Chapter 327.
Following the identification of pyruvate as the substrate of TYW1 a
Mössbauer and electron paramagnetic resonance (EPR) study was performed on
TYW126. This study confirmed that there is a second iron-sulfur cluster in TYW1 and that it is a 4Fe-4S cluster. The continuous wave EPR spectrum of reduced
TYW1 changed substantially upon the addition of both SAM and pyruvate. This indicates that both SAM and pyruvate are interacting with the 4Fe-4S clusters in
TYW1, with the authors hypothesizing that SAM interacts with one cluster, this has
25 been well established in many radical SAM enzymes by X ray crystallography28,29,
EPR30-33, Mössbauer31, and selenium X-ray absorption spectroscopy34,35, and the other cluster interacts with pyruvate. The Mössbauer spectrum of oxidized TYW1 is in support of pyruvate interacting with the unique iron of the second 4Fe-4S cluster. Based on this the authors proposed a mechanism in which pyruvate was coordinated to the unique iron of the second cluster26.
The subsequent steps in the biosynthesis of wyosine derivatives diverge depending upon which base is the end point in the pathway. The biosynthesis of the remaining bases in both eukaryotes and archaea will be discussed later in this chapter.
1.4 Wyosine biosynthesis in eukaryotes
To date there have been three wyosine derivatives, yW, OHyW, and imG7,8,11, found in eukaryotic tRNA. There have also been several other wyosine derivatives detected in eukaryotic tRNA, but they are present in low abundances as they are biosynthetic intermediates in the formation of the above three mentioned bases. Figure 1.5 shows the biosynthetic pathways of the three wyosine derivatives found in eukaryotes.
1.4.a Wybutosine (yW) biosynthesis
To date yW has been found in both plant and yeast tRNAPhe. The biosynthetic pathway of yW was determined in 2006 through the use of S. cerevisiae deletion strains in the same study that identified TYW117. That study
26 bases that are the end products oftheproductspathways end different the bases boxed.are are that 1.5 Figure
The biosynthetic pathway for wyosineTheforinpathwaybiosynthetic three eukaryotes. found derivativesThe
27 identified YML005w, YGL050w, and YOL141w as the genes responsible for the biosynthesis of yW, in addition to the above mentioned TRM5 and TYW1. The first two steps in the biosynthesis of yW, methylation of G37 to form m1G by TRM5 and formation of the tricyclic base imG-14 by TYW1, are common to the biosynthesis of all wyosine derivatives and were discussed above. The biosynthesis of yW has been reviewed recently with emphasis placed upon TYW1 in the two reviews36,37, please see Appendix A for one of the reviews.
1.4.a.i 7-aminocarboxypropyldemethylwyosine (yW-86)
Following the formation of the tricyclic base imG-14 by TYW1, the next enzyme in the pathway is 7-aminocarboxypropyldemethylwyosine (yW-86) synthase,
YML005w in S. cerevisiae (TYW2), as shown in Figure 1.517. Sequence alignments of TYW2 identified it as containing a SAM binding domain17 indicating SAM is most likely used during catalysis. Noma et al. reconstituted TYW2 activity in vitro using recombinently purified protein, tRNAPhe purified from a DTYW2 S. cerevisiae strain, and SAM, producing an RNA base with a m/z increase of 101
Da17. This is consistent with the addition of the a-amino-a-carboxypropyl group of
SAM forming yW-86.
In 2005 a separate group identified TYW2, which they named TRM12 (for tRNA methyltransferase), as being involved in yW biosynthesis. The group identified an increase in the level of imG-14 in a DTYW2 S. cerevisiae strain, along with a lack of yW in the tRNA extracted from this strain. They hypothesized that
TYW2 is a SAM dependent methyl transferase that could have been involved in
28 catalyzing the addition of one of the methyl groups or the a-amino-a- carboxypropyl group found in yW38, but they did not characterize the base produced by the enzyme.
Bioinformatics studies revealed TYW2 homologs in several archaeal genomes. In 2009 TYW2 from two of these archaeal strains, M. jannaschii and P. horikoshii, were assayed for activity in vitro and crystalized. Both of the TYW2 homologs are capable of producing yW-86 when provided with tRNAPhe extracted from a DTYW2 S. cerevisiae strain and SAM demonstrating that they are both yW-
86 synthases39. The crystal structures of TYW2 show that the protein has high structural similarity to class I SAM dependent methyltransferases such as TRM5.
The major structural difference between these enzymes and class I SAM dependent methyltransferases was the presence of a large bulky residue in the cavity that the methyl group of SAM is typically directed towards in methyl transferases. This causes the methyl group to be directed elsewhere and the a-amino-a- carboxypropyl group of SAM to bind correctly and be set up for transfer to the tRNA39.
1.4.a.ii 7-aminocarboxypropylwyosine (yW-72)
The next enzyme in the yW biosynthetic pathway is the SAM dependent methyltransferase TYW3, which produces 7-aminocarboxypropylwyosine (yW-
72).
TYW3 (YGL050w) is a SAM dependent methyltransferase. When recombinant TYW3 is combined with tRNAPhe from DTYW3 S. cerevisiae strain
29 and SAM, a tRNA is obtained with an increase in m/z of 14 Da. This indicates that
TYW3 methylate’s N-4 of yW-86 forming yW-7217.
1.4.a.iii Wybutosine (yW) – yeast
The final enzyme in the biosynthetic pathway is TYW4 which methylates the a-carboxy group and methoxycarbonylates the a-amino group of yW-72.
In the initial study that identified the genes involved in yW biosynthesis the activity of TYW4 (YOL141w) was reconstituted in vitro in the presence of SAM and tRNAPhe from a DYOL141w S. cerevisiae strain. Following the TYW4 reaction there were two new RNA species, yW and a species 58 Da less, indicating the presence of no methoxycarbonylate group in yW. The species 58 Da less (yW-58) was present in a very small amount suggesting that it is an intermediate, that eventually forms yW. The data led the authors to conclude that TYW4 was a SAM dependent methyltransferase that also methoxycarbonated the base forming yW17.
In 2009 the crystal structure of S. cerevisiae TYW4 was solved. The N- terminal domain of TYW4 is homologous to PPM1, a methyltransferase that methylates the C-terminus of protein phosphatase 2A40,41. The structure of the N- terminal domain of TYW4 also showed structural similarity to protein phosphatase
2A and other class I SAM dependent methyltransferases40,41. This suggests that methylation of the 7-aminocarboxypropylwyosine carboxy group occurs in the N- terminal domain of TYW4. The C-terminal domain of TYW4 does not show any obvious SAM binding pocket, but is homologous to adaptor proteins in cellular
30 signaling pathways. The authors proposed the C-terminal domain provides substrate specificity41.
To determine the source of the methoxycarbonyl group the authors tested
TYW4 for activity in the presence of just SAM and tRNAPhe from a DTYW4 S. cerevisiae strain. Following the incubation with TYW4 the tRNA contained yW instead of yW-72. This led the authors to conclude that either SAM or dissolved
CO2 in the reaction buffer was responsible for the formation of the methoxycarbonyl group. When the assay was performed in the presence of
13 NaH CO3 the yW produced had an increase in m/z of +1, indicating the incorporation of the labeled carbon. This showed that carbon dioxide is the source of the carbonyl in the methoxycarbonyl group and that SAM was the source of the methyl41.
The biosynthesis of yW in S. cerevisiae, and presumably in all other species, proceeds via the actions of five enzymes, TRM5, TYW1, TYW2, TYW3, and TYW4. These enzymes along with SAM, utilized by each enzyme in the following ways; whether it be as a methyl source in the case of TRM5, TYW3, and
TYW4; the source of dAdo• in the case of TYW1; or an a-amino-a-carboxypropyl group with TYW2; pyruvate, and carbon dioxide to form the very complex yW base from a genetically encoded guanosine.
1.4.a.iv Wybutosine (yW) – plants
A gene homologous to TYW2, TYW3, and the C-terminal domain of
TYW4 exists as a single fused gene in all sequenced plants. In addition to this gene,
31 homologs of TRM5, TYW1, and the PPM1 domain of TYW4 are also found in plants. It is hypothesized that these four genes are responsible for yW biosynthesis in plants, but it has yet to be experimentally shown17.
1.4.b Hydroxywybutosine (OHyW)
A wyosine derivative even more complex than yW, OHyW, is found in mammalian tRNA. When rat liver tRNA was initially sequenced O2yW was discovered, but subsequent sequencing detected OHyW and the O2yW was hypothesized to be a degradation product of OHyW11,12. OHyW is a hydroxylated version of yW and is installed by the enzyme TYW5.
TYW5 was identified initially by a bioinformatics study that discovered a previously uncharacterized globular domain that is fused to the C-terminus of a
TYW4 homolog in ascomycetes, choanoflagellates, and kinetoplastids42. The authors identified this protein as a member of the jumonji-related (JOR/JmjC) dioxygenase superfamily. JOR/JmjC dioxygenases utilize iron and 2-oxogluterate as cofactors, and until this point had only been characterized as protein modifying enzymes that demethylate histones via a hydroxylated intermediate43,44. This led the authors to propose TYW5 as the enzyme that catalyzes the formation of
OHyW42.
A 2010 study verified the activity of TYW5, both in vivo and in vitro45. The authors identified TYW5 as the enzyme that catalyzes the formation of OHyW in vivo by knocking down TYW5 in HeLa cells by RNAi. When the RNA from these cells was analyzed the levels of OHyW were decreased, while the levels of yW
32 were increased, compared to the control. This suggests TYW5 is responsible for hydroxylation of yW. In another experiment human TYW5 was introduced into S. cerevisiae via a plasmid and when TYW5 was expressed a small amount of OHyW was found in the RNA when it was analyzed via LC-MS. The human TYW5 gene was also introduced into a DTYW4 S. cerevisiae strain, the RNA extracted when
TYW5 was expressed in this strain was approximately 60% OHyW. This indicates that TYW5 preferentially modifies yW-72, not yW. Following the actions of
TYW5, TYW4 then forms the methoxycarbonyl group and methylates the carboxylate group. This was also confirmed in vitro using purified human TYW5 expressed in E. coli, tRNAPhe from a DTYW4 S. cerevisiae strain (contains yW-
Phe 72), or tRNA from S. cerevisiae (contains yW), 2-oxoglutarate, and Fe(II)SO4.
When the assay was carried out in the presence of tRNAPhe from the DTYW4 S. cerevisiae strain, undermodified hydroxywybutosine (OHyW*) containing tRNA was produced, but when wild type S. cerevisiae tRNAPhe was used as substrate no
OHyW was produced, showing that yW-72 containing tRNAPhe is the preferred substrate of TYW5. Incubation of the OHyW* containing tRNAPhe with S. cerevisiae TYW4, carbonate, and SAM lead to formation of tRNAPhe containing
OHyW45. Human TYW5 was crystalized in 2011 in the presence of nickel and 2- oxogluterate46. The biosynthetic pathway for the production of OHyW in humans is shown in Figure 1.5.
33 1.4.c Wyosine (imG)
The other wyosine derivative found so far in eukaryotic organisms is imG.
This derivative was originally found in T. utilis in 196847 and was structurally characterized in 1976 and found to be imG7. A BLAST search of the T. utilis genome reveals homologs for TRM5, TYW1, and TYW3, but no homologs for
TYW2 and TYW4. This is consistent with the presence of imG and not yW in T. utilis. A proposed biosynthetic pathway for wyosine is shown in Figure 1.5. The conserved wyosine biosynthetic enzymes TRM5 and TYW1 transform guanosine into imG-14. This would be followed by the SAM dependent methyltransferase
TYW3 methylating N-4 of imG-14, forming imG, which has yet to be shown experimentally. This is the simplest wyosine derivative found in eukaryotes.
1.5 Wyosine biosynthesis in archaea
The greatest diversity in wyosine derivatives exists in archaea with the following five different derivatives having been found in tRNAPhe; imG-14, imG, iso-wyosine (imG2), mimG, and yW-8648. Four of these wyosine derivatives, imG, imG2, mimG, and yW-86, are thought to be end products of biosynthetic pathways in archaea. The proposed biosynthetic pathways are shown in Figure 1.6.
All wyosine derivative biosynthesis proceeds via the m1G and imG-14 intermediates as described above in Chapters 1.3a and 1.3b in archaea, in the same manner as it does in eukaryotes.
34 pathways depending upon the organism. the pathwaysupon depending and guanosine of exception Figure 1.6
T he biosynthetic pathways for wyosine derivatives in archaea. base,Every with the N - ehlunsn ae on a ed rdcs f biosynthetic of products end as found are methylguanosine
35 1.5.a 4-demethylwyosine (imG-14)
The product of TYW1 is the tricyclic base imG-14, a wyosine derivative common to all wyosine derivative biosynthetic pathways. It is found as an intermediate, in eukaryotic organisms, during the biosynthesis of more complex derivatives, as described in section 1.3b. However, imG-14 has been detected as the only wyosine derivative present during sequencing of archaeal tRNA in M. burtonii49. This suggests that the minimal wyosine derivative, imG-14, is present as the wyosine derivative in that species.
Sequencing of archaeal tRNA in M. vannielii, M. maripaludis, M. jannaschii, M. igneus, and M. thermolithotrophicus also revealed the presence of imG-14, but it was not the only wyosine derivative detected50. In those species imG was also detected indicating that imG-14 may be present as a biosynthetic intermediate.
1.5.b Wyosine (imG)
The proposed biosynthetic pathway for imG was discussed in section 1.4c and it most likely involves homologs of TRM5, TYW1, and TYW3. A bioinformatics study revealed that homologs of TYW1 and TYW3 were present in
M. vannielii, M. maripaludis, M. jannaschii, M. igneus, and M. thermolithotrophicus leading to the hypothesis that imG is the wyosine derivative found in these species48. Sequencing of the tRNA from these organisms revealed the presence of both imG and imG-1450.
36 1.5.c 7-methylwyosine (mimG)
The first wyosine derivative discovered in archaeal tRNA was mimG, which was found in S. solfataricus in 198713. It was subsequently found in P. calidifontis tRNA48.
A bioinformatics study revealed the presence of three distinct homologs of
TRM5 in archaea, with some archaea harboring two homologs of TRM548. P. calidifontis only contained homologs for three putative wyosine biosynthetic enzymes; TRM5, TYW1, and TYW3; leading the authors to propose that the TRM5 homolog could be methylating both N1 of guanosine and C7 of imG-14. This was tested in vitro and was shown to be the case using TRM5 from P. abyssi and tRNA purified from a DTYW2 S. cerevisiae strain (contains imG-14) and [methyl-14C]-
SAM. When this assay was performed and the RNA was digested to 5′- monophosphate nucleosides and analyzed via two dimensional thin layer chromatography the radioactivity migrated in the same manner as mimG, showing that TRM5 was capable of methylating C7 of imG-1448. A proposed biosynthetic pathway for the synthesis of mimG is shown in Figure 1.6. The base imG-14 is synthesized as previously described by TRM5 and TYW1, which is followed by methylation of C7 by TRM5 to produce mimG48,51. The final base with a C7 methylation is imG2.
1.5.d Iso-wyosine (imG2)
The wyosine derivative imG2 is an isomer of imG with a methyl at C7 as opposed to N4 of the base. Unfractionated tRNA from M. igneus, M.
37 thermolithotrophicus, S. hydrogenophila, and S. solfataricus were all analyzed via
LC-MS and a base with the same mass of imG was detected, but with a different retention time. Comparison of the retention time of the unknown base with a synthetic standard led to the identity of the structure of imG252.
A proposed biosynthetic pathway for the formation of imG2 is shown in
Figure 1.6. The base imG-14 is synthesized as previously described by TRM5 and
TYW1. This is followed by methylation of N4 by TYW3 and finally methylation of C7 by TRM5, to give imG2.
1.6 Wyosine derivatives in mitochondria
In 2015 it was discovered that T. brucei contained two homologs of
TYW153. BLAST searches revealed this organism contained two homologs of
TYW1 and TYW3, along with a single homolog of TYW2 and TYW4. The TYW4 homolog had an extension that had homology to human TYW5. The two homologs of TYW1 differ in that one is a homolog of the eukaryotic enzyme (referred to as
TYW1L), which contains the N-terminal flavodoxin domain and the C-terminal radical SAM domain, while the other is a homolog of archaeal TYW1 (referred to as TYW1S) and only contains a radical SAM domain. The TYW1L protein was predicted to be cytosolic, which is consistent with all of the TYW1 proteins studied so far in eukaryotes53,54, while TYW1S was predicted to localize to the mitochondria53. Western blot analysis of the cytosolic and mitochondrion fractions confirmed that TYW1L was localized in the cytosol and TYW1S was localized in the mitochondria. The two TYW3 homologs showed the same localization pattern
38 as the two TYW1 homologs53. When the tRNA isolated from those fractions was analyzed for the presence of wyosine derivatives OHyW was found in the cytosolic fraction, while the mitochondrion fraction contained both OHyW and imG53.
Knocking down the TYW1L gene through RNAi resulted in the absence of OHyW in both the cytosolic and mitochondrion tRNA, while knocking down the TYW1S gene resulted in the absence of imG from the mitochondrion tRNA. This data suggests that TYW1L participates in the biosynthetic pathway leading to OHyW in the cytosol while TYW1S is involved in the biosynthesis of imG in the mitochondria53. This is the first instance of a wyosine derivative been detected in mitochondria.
1.7 Biological role of wyosine derivatives
The hydrophobic nature of yW and wyosine derivatives are thought to promote base stacking interactions and consequently limit flexibility of the anticodon loop55-57. There are certain disease states where wyosine derivatives are hypomodified such as in tumor cells and HIV infection.
In 1979 it was discovered that Ehrlich ascites tumor cells contain OHyW* instead of the expected OHyW in their phenylalanine encoding tRNA58.
Subsequent analysis of neuroblastoma cells revealed the presence of m1G instead of OHyW59. A later study found that hypomodified tRNAPhe was utilized preferentially in neuroblastoma cells, leading the authors to hypothesize that hypomodification of tRNAPhe may give a growth advantage to cancerous cells60.
39 When tRNAPhe from HIV infected cells was analyzed, it was discovered that the majority of the tRNA contained m1G, not OHyW as expected61. The Phe codon,
UUU, is located within the gag-pol frameshift site of HIV62,63 and would occupy the P site of the ribosome when the gag-pol frameshift site is been translated. The authors hypothesized that lack of the OHyW base would cause the tRNA to have more flexibility and thus increase the likely hood of a frameshift event occurring61.
A further study utilizing rabbit reticulocyte lysate and tRNAPhe with, or without the yW modification showed that frameshifting increased by 20% when yW was absent from the tRNA64.
The effect of yW modification on -1 frameshifting was further shown using a reported system in wild type, DTYW1, and DTYW2 S. cerevisiae strains65. The tRNA from the DTYW1 strain contained m1G, while that from the DTYW2 strain contained imG-14, and wild type contained yW. The results showed that frameshifting was greatest in the DTYW1 strain (tRNA contained m1G) and the lowest with the wild type (tRNA contained yW) with the DTYW2 frameshifting level between that of wild type and DTYW1 strain. The data shows that not only does the presence of a wyosine base in tRNAPhe have a large impact upon the frameshifting ability of that tRNA, but that the level of modification also has an impact with frameshifting decreasing as the base becomes more modified. This suggests that wyosine bases may have developed as a way to cope with frameshifting in phenylalanine encoding tRNA in eukaryotes and archaea65.
40 Chapter 2:
Radical S-adenosyl-L-methionine (SAM) enzymes
41 2.1 Common features of radical SAM enzymes
Members of the radical SAM super family are characterized by the presence of a cysteine rich motif22. There are over 100,00066 members of this super family and they catalyze a wide variety of reactions, playing a role in vitamin biosynthesis67, cofactor biosynthesis68,69, DNA repair70, RNA modification27,71, protein modification32,72, and glycyl radical generation73. Figure 2.1 shows the reactions catalyzed by TYW1 and the three most extensively studied radical SAM enzymes.
The radical SAM superfamily was initially characterized by the presence of a cysteine rich motif CxxxCxxC22. This sequence has subsequently been shown to deviate in some homologs, for example B. subtilis QueE contains the normal
74 75 CxxxCxxC motif while B. multivorans QueE contains a Cx14xCxxC motif , while in other cases all of the sequenced or characterized homologs have an abnormal cysteine motif such as ThiC67 and GenK76. The cysteine rich motif ligates a 4Fe-4S cluster via three of the four irons, with the fourth iron termed the “unique” iron. The unique iron has been shown to coordinate SAM via the amino and carboxy portions by X-ray crystallography28,29,73,75,77-83, EPR31-33, Mössbauer31, and selenium X-ray absorption spectroscopy34,35. The 4Fe-4S cluster cycles between the +1 and +2 oxidation states. When radical SAM enzymes are purified they are typically in the +2 state and require reductant to enter the catalytically active +1 state. In the catalytically active +1 state electron transfer from the cluster to SAM causes homolytic cleavage of the sulfonium 5′ ribose carbon bond forming the highly reactive intermediate dAdo•, and methionine.
42
Figure 2.1 This figure shows examples of the diverse reactions catalyzed by members of the radical SAM superfamily.
43 The highly reactive intermediate dAdo• subsequently goes on to abstract a hydrogen atom from a substrate, forming a substrate radical which will eventually go on to form product and 5′-deoxyadenosine (dAdoH)23, as is shown in Figure 2.2.
It has been documented in one enzyme, B12-independent glycerol dehydratase, that the sulfonium-g methionine bond is broken leading to formation of a thiomethyladenosyl radical that abstracts a hydrogen atom to form thiomethyladenosine, not dAdoH84.
2.2 Classes of radical SAM enzymes
There are three classes of radical SAM enzymes, class I, class II, and class
III radical SAM enzymes23. Class I radical SAM enzymes use SAM catalytically during the course of the reaction, as a cofactor. Following production of dAdo• and hydrogen atom abstraction to form dAdoH the substrate reabstracts the hydrogen atom from dAdoH to form dAdo• and the final product. The dAdo• reforms SAM with methionine and reduces the cluster in the process regenerating SAM and the cluster in the catalytically active +1 state. This only occurs in transformations that are redox neutral and the most extensively characterized class I radical SAM enzyme is lysine amino mutase23.
Class II radical SAM enzymes abstract a hydrogen atom from their substrate, which happens to be a glycine residue in a protein, generating a stable glycyl radical. This class of radical SAM enzymes use SAM as a substrate, generating dAdoH and methionine as products. The glycyl radical produced on the protein is stable and is used catalytically, regenerating at the end of each cycle. The
44
Figure 2.2 The catalytic cycle of the 4Fe-4S cluster in radical SAM enzymes showing the generation of 5′-deoxyadenosyl radical from SAM, which then goes on to abstract a hydrogen atom from substrate.
45 best studied class II radical SAM enzyme is pyruvate formate lyase activating enzyme, which was one of the four founding members of the radical SAM superfamily23.
Class III radical SAM enzymes make up the majority of radical SAM enzymes. These enzymes use SAM as a substrate producing dAdoH and methionine during the course of the reaction. Examples of this class of radical SAM enzymes include BioB, another of the founding members of the radical SAM super family, and TYW1, the focus of this dissertation23.
2.3 Radical SAM enzymes with auxiliary clusters
A subset of radical SAM enzymes have been shown to contain auxiliary Fe-
S clusters, and were recently reviewed85,86. The auxiliary clusters in the characterized radical SAM enzymes have been shown to be either 2Fe-2S83 or 4Fe-
4S clusters26,28,72,87-92. Radical SAM enzymes containing auxiliary clusters have been found to contain either one (two clusters total) or two (three clusters total) auxiliary clusters. The roles that the auxiliary cluster plays in the majority of cases is unknown, but they have been hypothesized to play a role in coordinating substrate26,28, source of sulfur atoms72,88,93,94, and play a role in electron transfer80,89,90.
2.3.a Sulfur insertion
The most characterized radical SAM enzymes with auxiliary clusters are those that catalyze the insertion of a sulfur atom or atoms. There are two different
46 types of sulfur insertion reactions catalyzed by radical SAM enzymes, those that insert a sulfur atom into a cofactor such as biotin or lipoic acid, or those that insert a methylthio group into both protein (RimO) and RNA (MiaB). The reactions catalyzed by these enzymes are shown in Figure 2.3.
2.3.a.i BioB (biotin synthase)
Biotin synthase (BioB) catalyzes the formation of biotin by the addition of a sulfur atom to dethiobiotin. When BioB was initially purified, it purified with 2 mol of iron and 2 mol of sulfide per mol of protein leading the authors to hypothesize it contained a 2Fe-2S cluster69. Subsequently the protein was found to contain an oxygen sensitive a 4Fe-4S and a 2Fe-2S cluster94,95, with the cluster identities confirmed in the X-ray crystal structure83.
Initial purifications of BioB produced protein that was capable of producing low amounts of product in the absence of an external sulfide source leading to speculation that it purifies with the sulfide source, or the protein itself is the
69,96 34 source . Utilizing BioB reconstituted in the presence of Na2 S or Na2Se biotin containing 34S97 or Se93 was produced demonstrating the source of sulfur as an Fe-
S cluster within the protein. UV-vis studies95 and Mössbauer studies98,99 have provided evidence for destruction of 2Fe-2S cluster during turnover, which is consistent with the 2Fe-2S cluster as the source of sulfur in biotin.
47
Figure 2.3 The sulfur insertion reactions catalyzed by radical SAM enzymes.
48 2.3.a.ii LipA (lipoyl synthase)
LipA is a radical SAM enzyme that catalyzes the insertion of two sulfur atoms into octanoic acid forming lipoic acid68,100. Studies revealed the presence of two 4Fe-4S clusters and also that it is capable of only a single turnover leading to the hypothesis that the cluster may supply the sulfurs100. Protein that was expressed in media containing 34S, caused the clusters to be labeled with 34S. The labeled protein was mixed with unlabeled protein and incubated with substrate. When the product was analyzed it was found that both the sulfur atoms had the same label, indicating that they were derived from the same protein101.
LipA cannibalizes its auxiliary cluster in the same manner as BioB to provide the sulfur atoms for lipoic acid and is capable of only one turnover.
2.3.a.iii Methylthiotransferases (MiaB and RimO)
There are two methylthiotransferases enzymes that have been characterized.
MiaB is involved in the transfer of a methylthio group to tRNA102, while RimO transfers a methylthio group to the S12 protein found in the ribosome103. Both of these enzymes contain two 4Fe-4S clusters32. Utilizing labeled SAM it was revealed that the protein is initially methylated at an acid labile site, which is then subsequently transferred to substrate72. The mechanism proposed for the two methylthiotransferases is initial methylation of a sulfide in the auxiliary 4Fe-4S cluster, which is subsequently transferred to the substrate tRNA or protein32,88.
49 Studies of the four enzymes BioB, LipA, RimO, and MiaB show that the auxiliary cluster of a radical SAM enzyme can be used as a source of sulfur in these cases. Other possible roles for the auxiliary clusters are discussed below.
2.3.b Coordination of substrate
The auxiliary clusters of some radical SAM enzymes have been proposed to coordinate substrate, either through evidence provided in an X-ray crystal structure28 or spectroscopic methods26,104.
2.3.b.i MoaA (molybdopterin cofactor biosynthesis)
The molybdopterin cofactor is derived from GTP and its biosynthesis involves the actions of 4 enzymes. The first step involves the radical SAM enzyme
MoaA and a second enzyme MoaC to produce the intermediate precursor Z, this is shown in Figure 2.4.
MoaA has been shown to contain two 4Fe-4S clusters, one of them the typical radical SAM cluster and the other an auxiliary cluster. Mutagenesis studies have shown that both clusters are required for activity. The cysteines in the radical
SAM cluster sequence of the A. nicotinovorans MoaA gene were each mutated to serine and the resulting protein produced could not rescue molybdopterin cofactor biosynthesis in an in vivo complementation assay in a MoaA knockout E. coli strain105. Another study by the same group mutated the three cysteines to serines in the second cysteine rich motif and also found the resulting protein unable to
50
Figure 2.4 The conversion of GTP to precursor Z by the actions of two enzymes, the radical SAM enzyme MoaA and MoaC.
51 rescue molybdopterin cofactor biosynthesis in a similar in vivo complementation assay, showing they are required for activity106.
Initial purifications of MoaA obtained a brown protein that was EPR active, in the presence of dithionite, that contained four mol of iron and three mol of acid labile sulfide per mol of protein leading to the conclusion that MoaA is an Fe-S protein106. A subsequent study of anaerobically purified MoaA with cysteine to serine mutations of each cluster binding motif revealed that each of the three cysteines coordinated a 4Fe-4S cluster107. The presence of two 4Fe-4S clusters was confirmed via X-ray crystallography28,108.
The crystal structure of MoaA in complex with GTP revealed an interaction between it and the auxiliary cluster. The N1 nitrogen and exocyclic amine of the guanine ring bind to the unique iron of the auxiliary cluster, with the keto at position six interacting with a residue in the protein28. An electron nuclear double resonance
EPR study of MoaA in complex with GTP revealed the interaction of a single nitrogen with the cluster. In order to determine which nitrogen, either N1 or the exocyclic amine, is interacting with the cluster the authors performed the same experiment using inosine triphosphate. Inosine triphosphate lacks the exocyclic amine, so if the spectrum obtained was the same as that of MoaA in complex with
GTP then the evidence would point to an interaction with the N1 nitrogen. The spectra of MoaA in complex with GTP and inosine triphosphate are the same indicating that the interaction is between N1 and the auxiliary cluster104. The interaction of GTP with the auxiliary cluster of MoaA provides evidence that the auxiliary cluster can coordinate substrate and was unprecedented at the time.
52 2.3.c Peptide and protein modifying radical SAM enzymes
A large class of radical SAM enzymes containing multiple Fe-S clusters that modify peptides have been identified109. A domain has been identified within these proteins that has been termed a SPASM domain after the first biochemically characterized members of the members AlbA, PqqE, anSME, and MftC110. The following section will review what is known about roles of the auxiliary clusters in these enzymes.
2.3.c.i AlbA (subtilosin A biosynthesis)
Subtilosin A is a ribosomally synthesized post translationally modified peptide that shows antimicrobial activity111. AlbA was identified as a gene within the sho-alb locus, required for subtilosin A biosynthesis112,113. The structure of subtilosin A revealed the presence of three thioether crosslinks between three cysteines and the α carbons of two phenylalanine and one threonine residues114,115.
In vitro studies of AlbA revealed that it catalyzed the formation of all three thioether crosslinks116.
Iron and sulfide analysis of the wild type protein revealed approximately 8 mol of iron and sulfide per mol of protein reveling the presence of two clusters. The cysteine residues ligating the radical SAM cluster were each mutated to alanine and a variant where all three mutated to alanine was also created. These mutants were unable to produce both modified peptide and reductively cleave SAM to form dAdoH. EPR analysis of these mutants also revealed a spectrum that was consistent with the presence of an Fe-S cluster, and the iron and sulfide analysis was consistent
53 with the presence of one 4Fe-4S cluster. A triple mutant in which three additional conserved cysteines, identified via sequence alignments, were mutated to alanine was created. EPR analysis, along with iron and sulfide analysis, of this mutant was consistent with the presence of only one 4Fe-4S cluster, and it was assayed for activity it was both unable to form dAdoH and modified peptide116.
In order to determine if substrate is interacting with the auxiliary 4Fe-4S cluster, the authors studied it via UV-vis spectroscopy. The absorbance spectrum of AlbA in the presence of substrate shows a decrease in absorbance at 310 nm when compared with that of AlbA only116. The authors also constructed a truncated substrate in which the leader sequence was removed, studies have shown that the leader sequence is required for recognition by modifying enzymes, and when this was combined with AlbA there was no change in absorbance. When the triple mutant, where the cysteine residues thought to coordinate the auxiliary cluster, was combined with substrate there was also no decrease in absorbance as seen in wild type. This led the authors to hypothesize that the role of the auxiliary cluster in
AlbA is for substrate coordination and they proposed a mechanism in which the auxiliary cluster coordinates substrate and accepts an electron during the course of the reaction116.
A subsequent study by another group identified another four conserved cysteines via a homology model with anSME. The authors mutated the four cysteine residues to alanine residues and also made a variant in which all four were mutated to alanine residues. When these mutants were assayed for activity it was found that the only two of the single mutants could make small amounts of product.
54 When the UV-vis spectra were compared with that of wild type, the variant with the four cysteine residues mutated had a spectrum that differed from that of wild type indicating the loss of a cluster. This led the authors to hypothesize that there were three 4Fe-4S clusters in AlbA and they proposed that the three clusters played a role in electron transfer during the reaction117.
2.3.c.ii SkfB (sporulation killing factor biosynthesis)
The sporulation killing factor is a ribosomally assembled and post translationally modified peptide produced by B. subtilis118,119. The structure of sporulation killing factor revealed it is a circular peptide with one thioether bond linking a cysteine residue with the α carbon of a methionine residue120.
Sequence alignments revealed two conserved cysteine motifs, the typical radical SAM motif and an additional C-terminal cysteine motif. EPR analysis, along with iron and sulfide analysis (8 mol of each per mol of protein) of wild type was consistent with the presence of at least one and most likely two 4Fe-4S clusters.
Site directed mutagenesis of each cysteine motif, to alanine motif, produced mutants that were unable to produce modified peptide, but still retained one 4Fe-
4S cluster according to EPR and iron and sulfide analysis. The variant with the radical SAM cluster motif mutated to alanine was unable to cleave SAM and produce dAdoH, however when the auxiliary cluster motif is mutated to alanine the enzyme is still able to cleave SAM and form dAdoH121.
55 The authors hypothesized that the cluster plays a similar role as that in AlbA with it playing a role in binding and coordinating the substrate and may subsequently play a role in electron transfer to remove an electron efficiently from the product/substrate121.
2.3.c.iiiThnB (thurincin H maturation)
Another ribosomally synthesized post translationally modified peptide with antimicrobial properties is thurincin H122. The NMR structure of thurincin H revealed the presence of four thioether bonds between two threonine, one asparagine, and one serine residues and four cysteines123.
The radical SAM enzyme ThnB was purified and shown to catalyze the formation of the four thioether bonds in vitro. The EPR spectrum, along with iron and sulfide analysis of the wild type enzyme was consistent with the presence of two 4Fe-4S clusters. A triple alanine variant in which the cysteine residues that coordinate the radical SAM Fe-S cluster were mutated, was found to be unable to produce either modified peptide and dAdoH. The EPR, along with iron and sulfide analysis of this variant was consistent with the presence of one 4Fe-4S cluster. In order to probe the location of the auxiliary cluster a variant in which the three cysteine residues hypothesized to coordinate the auxiliary 4Fe-4S cluster were mutated to alanine. This protein was unable to modify peptide, but it was able to produce dAdoH, showing that the auxiliary cluster is required for turnover.
Moreover, the protein retained UV-visible and EPR spectral features consistent with the presence of a 4Fe-4S cluster124. The authors hypothesized that this enzyme
56 may have a mechanism similar to that of AlbA116, but did not provide any evidence or hypothesize a role for the auxiliary cluster beyond this124.
2.3.c.iv Tte1186 (SCIFF peptide modifying enzyme)
A recently characterized member of the radical SAM superfamily with a
SPASM domain is Tte1186, which modifies the six cysteines in forty-five (SCIFF) family of peptides92,109. Tte1186 catalyzes the formation of a thioether bond between a conserved cysteine and a threonine residue in its cognitive SCIFF peptide substrate92.
Site directed mutagenesis was used to identify three sets of cysteine residues that coordinate three separate 4Fe-4S clusters92. The three cysteine residues that make up the radical SAM motif were all mutated to alanine to produce a
AxxxAxxA motif, iron and sulfide analysis of this variant was consistent with an additional two 4Fe-4S clusters. When this variant was assayed for the cleavage of
SAM via the production of dAdoH, it did not produce dAdoH as expected and was also unable to produce modified peptide92.
There are an additional seven cysteines conserved in homologs of this protein. The authors hypothesized that these coordinate two additional 4Fe-4S clusters, with one of them coordinated by three cysteines and other four cysteines.
Variants in which the cysteines hypothesized to ligate each auxiliary cluster were mutated to alanine were consistent with the presence of two clusters. EPR performed upon the three variants was also consistent with the lack of an Fe-S cluster in each variant and three distinct clusters in wild type. When these variants
57 were assayed for activity they were unable to make product, but were able to cleave
SAM forming dAdoH, suggesting the clusters are important for activity, but not for cleaving SAM92.
There was no evidence for the role of the auxiliary clusters in this study, but the authors hypothesized that they could play a role in electron transfer. The mechanism proposed for this transformation requires the loss of an electron92, which could be transferred via the two clusters to an external electron acceptor.
2.3.c.v PqqE (pyrroloquinoline quinone cofactor biosynthesis)
Pyrroloquinoline quinone cofactor is found in bacteria where it functions as a redox cofactor125. Five gene products were identified as required for pyrroloquinoline quinone cofactor production, with one of them encoding a radical
SAM enzyme (PqqE) and another a peptide126. The cofactor has been hypothesized to be derived from a glutamate and tyrosine residue127, which is encoded on a precursor peptide126.
PqqE is a radical SAM enzyme that has been shown to form a form a cross link between a glutamate and tyrosine residue in the precursor peptide128. PqqE expressed and purified under anaerobic conditions contain 10 and 7 mol of iron and sulfide, respectively, suggesting the presence of two 4Fe-4S clusters. The UV-vis spectrum is also consistent with the presence of two 4Fe-4S clusters as the absorbance at 410 nm is twice that expected for a single 4Fe-4S cluster129.
Subsequent iron and sulfide analysis of PqqE following reconstitution identified the presence of 13 mol of iron and 12 mol of sulfide per mol of protein.
58 Sequence alignments reveal the presence of ten conserved cysteines leading to the hypothesis that PqqE binds 4Fe-4S clusters. The roles of the clusters are unclear and the authors propose a role in which they remove an electron during the course of the reaction, providing a safe route for the disposal of a radical intermediate128.
2.3.c.vi StrB (streptide biosynthesis)
The StrA gene encodes a small peptide that is post translationally modified to form a cyclic peptide with a crosslink between a lysine and tryptophan residue130,131 that plays a role in quorum sensing130. StrB, a radical SAM enzyme, was shown in vitro to catalyze the formation of the crosslink between the lysine and tryptophan residues131.
Based upon sequence alignments with anSME it was hypothesized that StrB contains at least one auxiliary cluster. Iron and sulfide analysis of the wild type enzyme revealed nine mol of iron and seven mol of sulfide per mol of protein. A double mutant variant in which two of the cysteines hypothesized to coordinate the auxiliary cluster were mutated to alanine residues. This variant was found to be unable to modify peptide. Iron and sulfide analysis of this variant revealed 4 mol of iron and sulfide per mol of protein, which is consistent with the presence of one
4Fe-4S cluster131.
A mechanism for this transformation was proposed in which the auxiliary cluster accepts an electron during the course of the reaction131, which is similar to the role proposed in AlbA116,117.
59 2.3.c.vii MftC (mycofactocin biosynthesis)
A bioinformatics study revealed the presence of a gene cluster, containing three genes, that was found in at least 336 genomes109. This cluster is hypothesized to produce the as yet uncharacterized cofactor mycofactocin109. One of the genes encodes a hypothesized peptide, MftA, and one a radical SAM enzyme, MftC132.
The radical SAM enzyme was shown to catalyze the reductive decarboxylation of the C-terminus of the peptide87,132, interestingly this is the first characterized peptide modifying radical SAM enzyme that does not produce an internal crosslink.
Initial iron and sulfide analysis by two groups have revealed levels of both iron and sulfide consistent with the presence of two clusters, with both groups detecting approximately 10 mol of iron and 8 mol of sulfide per mol of protein87,132.
An alignment of the MftC with anSME revealed the presence of an additional four conserved cysteines that bind an auxiliary cluster in anSME. This led the authors to propose the presence of two clusters87. The auxiliary cluster may play a role in quenching a radical intermediate during the course of the reaction132, as has been proposed in AlbA116,117.
2.3.d Radical dehydrogenase subfamily
A subset of radical SAM enzymes containing auxiliary clusters catalyze the two electron reduction of a serine or cysteine residue, in the case of the anaerobic sulfatase maturation enzymes, or a hydroxyl group in the case of BtrN.
60 2.3.d.i AtsB (anaerobic sulfatase maturation enzyme)
Sulfatases catalyze the cleavage of organosulfate esters within cells133. One group of sulfatases contain a formyl-glycyl residue that is derived from a serine residue, in the sulfatase, and its transformation is catalyzed by the radical SAM enzyme AtsB134-136.
AtsB is capable of modifying a serine residue in an 18 amino acid peptide whose sequence is identical to that of AtsA (immature sulfatase) to a formyl glycine residue and cleaving SAM to form dAdoH. The reconstituted wild type protein contained 12 mol of iron and 10 mol of sulfide per mol of protein. The Mössbauer spectrum of this protein was consistent with the presence of three 4Fe-4S clusters.
The presence of three 4Fe-4S clusters is collaborated with sequence alignments in which ten conserved cysteines are present. A variant of AtsB in which the cysteines ligating the radical SAM motif were all mutated to alanine residues was inactive and bound 7 mol of iron and 8 mol of sulfide per mol of protein, which is consistent with presence of two auxiliary clusters91.
The authors proposed a mechanism in which the serine residue was initially activated by binding to the unique iron of one of the auxiliary Fe-S clusters. This is followed by one of the auxiliary clusters accepting an electron from the substrate, and subsequently transferring it to an external acceptor91.
61 2.3.d.ii anSMEcpe (cysteine anaerobic sulfatase maturation enzyme)
Another example of a sulfatase maturation enzyme is anSMEcpe. This enzyme transforms a cysteine residue into the formylglycine residue required for sulfatase activity137.
Characterization of the iron and sulfide content of this protein was consistent with the presence of three 4Fe-4S clusters90, as was found in AtsB91.
Additional mutagenesis of each one of the motifs thought to ligate each of the two auxiliary clusters led to variants containing iron and sulfide consistent with the presence of two 4Fe-4S clusters in both cases90. The interesting discovery in this study was that the protein is capable of undergoing multiple turnovers following the initial reduction. If a flavodoxin/flavodoxin reductase reducing system is used, following the initial reduction of the radical SAM Fe-S cluster the enzyme is able to recycle the electron generated during catalysis, probably transferred via the two auxiliary clusters to the flavodoxin/flavodoxin reductase system where it is used to reduce the radical SAM cluster and allow the catalytic cycle to proceed once more.
This is the first report of the clusters transferring electrons in this manner and provides evidence for one of their purposes in the protein90. This hypothesis was supported by with the X-ray crystal structure of anSMEcpe, in which one of the auxiliary clusters was found to be 8 Å away. It was hypothesized that the electron would transfer to this cluster, and from there to the second auxiliary cluster which is 13 Å away. From this cluster it was hypothesized the electron could be transferred to an external acceptor such as the flavodoxin/flavodoxin reductase system and from there reduce the radical SAM cluster again81.
62 Based on this data it can be hypothesized that the auxiliary clusters play a role in electron transfer, at least in the case of anSMEcpe, where the substrate is too far from the cluster in interact81.
2.3.d.iii BtrN (2-deoxy-scyllo-inosamine biosynthesis)
BtrN is a radical SAM enzyme that catalyzes the two electron reduction of a hydroxyl group to a ketone creating 3-amino-2,3-dideoxy-scyllo-inosone from 2- deoxy-scyllo-inosamine138. Initial characterization of the enzyme identified that the enzyme used SAM catalytically and hypothesized that an intermediate substrate radical was donated back to the 4Fe-4S cluster that cleaves SAM during the course of the reaction restoring the cluster to an active oxidation state. This study only identified the presence of one 4Fe-4S cluster138.
A subsequent study quantified the levels of iron and sulfide in the reconstituted protein and found 7.7 and 8.8 mol of iron and sulfide, respectively, per mol of protein. These numbers are consistent with the presence of two 4Fe-4S clusters. When the signature radical SAM binding motif cysteines were mutated to alanine residues, the resulting protein contained 3.7 and 4.6 mol of iron and sulfide, respectively, per mol of protein which is consistent with the presence of one auxiliary cluster. A mechanism was proposed in which the auxiliary cluster binds substrate and accepts an electron from a substrate radical during the course of the reaction, possibly transferring it back to the radical SAM 4Fe-4S cluster89.
The crystal structure of BtrN revealed that the substrate does not interact with the auxiliary cluster. Instead, it was hypothesized that the auxiliary cluster
63 accepts an electron from the substrate radical species during the course of the reaction and transfers it to an external electron acceptor, as was proposed in anSMEcys80. An electrochemical study of the two clusters in BtrN revealed that the auxiliary cluster (-765 mV) has a much lower potential than the radical SAM cluster
(-510 mV). The authors proposed the auxiliary cluster accepts an electron from the substrate radical and can then transfer it to either the radical SAM cluster or an external electron acceptor139.
In the case of both BtrN and anSMEcpe a mechanism has put forward were the clusters transfer a radical generated during the reaction to an external electron acceptor, as opposed to binding substrate80,89,90,139. In the case of anSMEcpe, there is evidence that this hypothesis is correct as that enzyme is capable of multiple turnovers after an initial reduction of the clusters, when supplied with a flavodoxin/flavodoxin reductase reducing system, which may transfer the electron from the auxiliary cluster back to the radical SAM cluster90.
2.4 Conclusion
A wide range of radical SAM enzymes contain auxiliary Fe-S clusters.
Some of the roles of these clusters, such as in the case of sulfur inserting enzymes are well established. However, a large percentage of the auxiliary clusters found in radical SAM enzymes do not have a well-established role. In the majority of these cases the cluster is hypothesized to accept an electron during the course of the reaction and safely remove it from the active site of the enzyme to an external electron acceptor or to bind substrate.
64 Chapter 3:
Pyruvate is the source of the two carbons that are required for conversion of
N-methylguanosine to the imidazoline tricyclic base of 4-demethylwyosine
This work has been published previously and is reproduced with permission from:
Young, A. P., and Bandarian, V. (2011) Pyruvate is the source of the two carbons that are required for conversion of N-methylguanosine to the imidazoline tricyclic base of 4-demethylwyosine, Biochemistry 50, 10573-10575.
The manuscript was written by A. P. Young and V. Bandarian. All experiments were designed by A. P. Young and V. Bandarian. All experiments were performed by A. P. Young.
65 3.1 Abstract
TYW1 catalyzes the condensation of N-methylguanosine with two carbon atoms from an unknown second substrate to form 4-demethylwyosine, which is a common intermediate in the biosynthesis of all of the hypermodified RNA bases related to wybutosine found in eukaryal and archaeal tRNAPhe. Of the potential substrates examined, only incubation with pyruvate resulted in formation of 4- demethylwyosine. Moreover, incubation with C1, C2, C3, or C1,2,3-13C-labeled pyruvate showed that C2 and C3 are incorporated while C1 is not. The mechanistic implications of these results are discussed in the context of the structure of TYW1.
3.2 Introduction
Of the 151 modifications that have been documented in RNA, 92 occur in tRNA and many are conserved in all domains of life2. The biosynthetic pathways leading to these modifications are often quite complex and ripe with novel chemistries.
yW and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea9. yW and its derivatives are installed in a series of reactions
(see Figure 3.1) that all require SAM. The first step of the pathway is methylation
1 of N-1 of G37 to generate m G, which is converted to the tricyclic ring of imG-14 in a reaction catalyzed by TYW1. imG-14 is converted to yW by the successive actions of TYW2, TYW3 and TYW4. TRM5 and TYW1 homologs are common to all organisms containing yW and yW derivatives. Thus imG-14 is a common intermediate to yW and yW derivatives in all organisms that produce the
66
Figure 3.1 Biosynthetic pathway of yW. G at position 37 is transformed to imG- 14 through the actions of TRM5 and TYW1, which is subsequently converted to yW through the actions of TYW2, TYW3, and TYW4. The naming conventions used throughout this paper are for the free nucleoside.
67 hypermodified base. Evidence for the biosynthetic pathway has accumulated through gene knock-out studies in yeast17.
TYW1 has been classified as a member of the radical SAM superfamily22 on the basis of a conserved CxxxCxxC motif, which provides 3 Cys thiolate ligands to form a catalytically essential [4Fe-4S]+2/+1 cluster. The cluster presumably binds and reductively cleaves SAM to generate dAdo• for radical-mediate transformation, as is the case for other radical SAM proteins23. In TYW1, the first step is presumed to be abstraction of a hydrogen atom from the methyl group of m1G to initiate radical-mediated condensation with a two carbon-donating second substrate, leading to formation of imG-14. The identity of the second substrate has remained elusive, thus hampering mechanistic studies of TYW1, for which several high- resolution structures are known24,25.
We have established a biochemical assay to study the source of the two carbon moiety that is required for the conversion of m1G to imG-14. For reasons of ease of purification and stability, all of our experiments were carried out with the Methanococcus jannaschii homologs of TRM5 and TYW1. The biochemical function of TRM5, conversion of G to m1G, has been documented140; therefore,
TRM5 could be utilized to generate m1G-containing tRNAPhe in situ.
3.3 Results and discussion
The recombinant M. jannaschii homolog of TYW1 was purified anaerobically and reconstituted with Fe and S. The reconstituted protein was brown and had an absorbance spectrum that displayed a characteristic shoulder at 400 nm
68 (Figure 3.2). The m1G-containing tRNAPhe (MJ_t16) was prepared by in vitro run off transcription.
The assays were carried out anaerobically in the presence of tRNAPhe,
TYW1, TRM5, and SAM. The reaction mixtures contained all components that have been shown to be required for activity of TRM5 (SAM and Mg2+)140. In addition, sodium dithionite was included to reduce the radical SAM cluster of
TYW1. Incubations were carried out for 12 h at 60 °C, following which reactions were quenched and the RNA was extracted digested to nucleosides, and analyzed via LC-MS as described previously141. The modified bases have distinct retention times and can be detected readily in the extracted ion chromatograms at m/z 298
(m1G) or 322 (imG-14); these correspond to the [M+H+] ions of m1G and imG-14, respectively.
Four compounds, acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate, were tested as possible two carbon-sources. Of these only incubation in the presence of pyruvate led to a reduction of the m1G peak and appearance of imG-
14 (m/z 322) in the extracted ion chromatogram (Figure 3.3). We found that methyl viologen, while not essential, stimulated activity; therefore, it was included in all subsequent reactions. Control experiments established that imG-14 does not form in the absence of TYW1 (Figure 3.4). Control reactions excluding SAM could not be carried out because it is a substrate for TRM5, which is utilized for synthesis of the m1G containing tRNAPhe in situ.
To gain additional insights into the mechanism by which pyruvate, a three- carbon compound, can be the source of the two carbons in the tricyclic system of
69
Figure 3.2 UV-visible spectrum of reconstituted TYW1. The trace showing the full spectrum was obtained with protein at 26 µM and the one highlighting the features due to the iron-sulfur cluster was obtained with 51 µM protein. The broad shoulder from 300 to 500 nm is characteristic of [Fe-S] clusters.
70
Figure 3.3 The extracted ion chromatograms showing the m1G peak (m/z 298), at an elution time 37-38 minutes, and imG-14 peak (m/z 322) at an elution time of 48 minutes. The peak due to m1G decreases in the presence of pyruvate, with the concomitant appearance of imG-14.
71
Figure 3.4 Extracted ion chromatograms showing that TYW1 is required for the formation of imG-14. In the absence of TYW1, only the m1G peak (m/z 298) at an elution time of 35 minutes is observed. In its presence, the m1G peak is reduced in favor of imG-14 (m/z 322) which appears at an elution time of 45 minutes.
72 yW, the experiments were repeated with several 13C-labeled analogs. Extracted ion chromatographs at m/z of imG-14 (m/z 322) and at +1 and +2 are shown in Figure
3.5. In the presence of unlabeled pyruvate, the extracted ion chromatograms show a peak at the expected m/z 322. Interestingly, when the reaction was carried out in
13 the presence of [1- C1]-pyruvate, an identical set of extracted ion chromatographs were obtained, indicating loss of C1. When the assays were carried out with [2-
13 13 C1]- or [3- C1]-pyruvate, however, the m/z 322 disappeared and a peak appeared in the extracted ion chromatogram at m/z 323. This suggests that both C2 and C3 of pyruvate are incorporated into the tricyclic ring of yW. Consistent with these
13 labeling patterns, when the reaction was carried out in the presence of [1,2,3- C3]- pyruvate, only a +2 shift to m/z 324 was observed. These results clearly demonstrate that the C2 and C3 of pyruvate are the source of the two carbon atoms that are required for synthesis of the tricyclic ring of yW. Note that the smaller peaks in the chromatograms at +1 are from the expected natural abundance isotopes of imG-14, which is expected to be ~16% of the intensity of the [M+H+] peak in each instance. We also carried out LC-MS/MS runs where unlabeled or singly labeled imG-14 (m/z 322 or 323 respectively) was trapped and fragmented; in each
+ case the [MB2 ] ion corresponding to the base carries the appropriate m/z, consistent with previous imG-14 fragmentation studies17,52.
Interpretation of the pyruvate labeling results with TYW1 can be carried out in the context of the two structures and mutagenesis results that are available in the literature24,25. Six Cys residues are conserved in TYW1, three of which occur in the radical SAM signature sequence (C62xxxC66xxC69, M. jannaschii numbering).
73
Figure 3.5 Extracted ion chromatograms showing incorporation of C2 and C3 of pyruvate into 4- demethylwyosine.
74 The thiolate sidechains of these residues would be expected to bind three of the metal ions in the [4Fe-4S] cluster; the fourth iron is presumably coordinated to the a-amino and a-carboxylate of SAM, as has been shown previously for other radical
SAM proteins142. The radical SAM cluster is located on one side of a positively charged putative active site cleft, where tRNA is proposed to bind and flip the m1G precursor for modification24. The three additional conserved Cys residues (Cys26,
Cys39, and Cys52) are located adjacent to the substrate binding site on the opposite side of the cleft from the SAM binding cluster. It has been proposed that these Cys residues could also form a [4Fe-4S] cluster. A conserved Lys residue (K41) adjacent to the second set of conserved Cys residues is also required for activity based on in vivo studies25. When one considers where pyruvate would bind, it seems reasonable to propose that the pyruvate binding site is on the same side as the Lys, which it may form a Schiff base with to facilitate the chemistry.
Our working model for the mechanism of TYW1, shown in Figure 3.6 is as follows. We propose that the conserved Lys residue forms a Schiff base with pyruvate. Reductive cleavage of SAM leads to formation of dAdo•, which either directly or perhaps through a protein side chain, propagates the radical to the m1G generating a substrate radical. Addition of the radical to C2 of pyruvate followed by homolytic scission of the C1–C2 bond would generate an intermediate, which through transimination and subsequent deprotonation, forms imG-14.
Decarboxylations, such as that proposed here, have been proposed in the reaction of pyruvate formate-lyase143 and coproporphyrinogen synthase (HemN)144. The resulting formyl radical can either acquire an H atom or undergo reduction and
75
Figure 3.6 Putative mechanism for the transformation of N-methylguanosine to 4-demethylwyosine.
76 protonation to produce formate. Alternatively, oxidation of the formyl radical would generate CO2. At this point, given the uncertainty about the involvement of enzyme-based radicals and the nature of the structure formed by the second set of conserved Cys residues, it is difficult to differentiate among these many possibilities. We note that while a Lys residue would be desirable, the transformations proposed in Figure 3.6 could also occur with pyruvate alone; however, a Schiff base would provide an attractive electron sink to stabilize the intermediates. The M. jannaschii protein used in these studies lacks a flavin mononucleotide binding domain that appends the protein in higher organisms; its absence from archaeal proteins may indicate that it has an alternative mechanism for cluster reduction.
In summary, we have identified pyruvate as the second substrate for TYW1 in the production of imG-14 on the pathway to yW. We have successfully reconstituted activity in vitro using isotopically labeled pyruvate. The isotope labeling patterns unambiguously show that C2 and C3 of pyruvate are incorporated into the tricyclic base, whereas C1 is lost. These observations, when taken with the
X-ray crystal structures, conserved residues, and mutagenesis data in the literature17,24,25,48 provide support for the model proposed here, which will be useful in directing future mechanistic studies of the fascinating transformation catalyzed by TYW1.
77 3.4 Materials and methods
Cloning of TRM5 and TYW1
Codon-optimized genes corresponding to the TRM5 (MJ_0883) and TYW1
(MJ_0257) of Methanocaldococcus jannaschii were obtained from GenScript
(Piscataway, NJ). The gene sequence and the corresponding protein sequence are shown in Figures 3.6 and 3.7. The genes were excised from the pUC57 constructs obtained from GenScript and cloned into NdeI and HindIII sites of pET29a (TRM5) and pET28a (TYW1).
Expression of recombinant TRM5
Plasmid containing TRM5 was transformed into BL21 (DE3) competent cells. For expression, one colony was grown overnight in 0.1 L of Lenox broth
(LB) containing 34 µg/mL kanamycin at 37 °C. The overnight culture was distributed among six 2.8 L Fernbach flasks containing 1 L LB and 34 µg/mL of kanamycin. The cells were grown to OD600 ~ 0.5; expression was induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.5 mM. Cells were harvested by centrifugation at 4,000 xg and frozen at – 80 °C.
Expression of recombinant TYW1
To ensure proper assembly of the iron-sulfur cluster in this protein, the pET28 plasmid containing the gene was co-transformed into BL21 (DE3) along with pDB1282 plasmid (from Dennis Dean, Virginia Tech, Blacksburg, VA,
78 DNA sequence
CATATGCCGCTGTGCCTGAAAATTAACAAAAAACATGGCGAACAGACCCGTCGCATTCTGATC GAAAACAATCTGCTGAACAAAGATTACAAAATCACGAGCGAGGGTAACTATCTGTACCTGCCG ATTAAAGATGTTGACGAAGATATCCTGAAATCTATCCTGAACATCGAATTCGAACTGGTGGAC AAAGAACTGGAAGAAAAGAAAATTATCAAAAAACCGAGTTTCCGTGAAATCATCTCCAAAAAA TACCGCAAAGAAATCGATGAAGGCCTGATCAGCCTGTCTTACGACGTGGTTGGTGATCTGGTG ATTCTGCAAATCAGTGACGAAGTTGATGAAAAAATCCGTAAAGAAATCGGCGAACTGGCATAT AAACTGATTCCGTGCAAAGGCGTTTTTCGTCGCAAATCCGAAGTCAAGGGTGAATTCCGTGTG CGCGAACTGGAACATCTGGCTGGCGAAAACCGTACCCTGACGATTCACAAAGAAAATGGTTAT CGCCTGTGGGTTGATATCGCGAAAGTCTACTTTTCACCGCGTCTGGGCGGTGAACGTGCACGT ATTATGAAAAAAGTTTCGCTGAACGACGTCGTGGTTGATATGTTTGCGGGCGTGGGTCCGTTC AGCATTGCGTGTAAAAACGCGAAGAAAATTTACGCAATTGATATCAATCCGCATGCTATTGAA CTGCTGAAGAAAAACATCAAACTGAACAAACTGGAACACAAAATCATCCCGATTCTGTCTGAC GTCCGTGAAGTCGATGTGAAAGGCAACCGCGTGATCATGAATCTGCCGAAATTTGCGCATAAA TTCATTGACAAAGCCCTGGATATCGTGGAAGAAGGCGGTGTTATTCACTATTACACCATCGGT AAAGACTTCGATAAAGCCATCAAACTGTTCGAGAAAAAATGCGACTGTGAAGTTCTGGAAAAA CGTATCGTCAAAAGCTATGCACCGCGCGAATACATTCTGGCTCTGGATTTCAAAATCAATAAA AAATAAAAGCTT Protein sequence
MPLCLKINKKHGEQTRRILIENNLLNKDYKITSEGNYLYLPIKDVDEDILKSILNIEFELVDK ELEEKKIIKKPSFREIISKKYRKEIDEGLISLSYDVVGDLVILQISDEVDEKIRKEIGELAYK LIPCKGVFRRKSEVKGEFRVRELEHLAGENRTLTIHKENGYRLWVDIAKVYFSPRLGGERARI MKKVSLNDVVVDMFAGVGPFSIACKNAKKIYAIDINPHAIELLKKNIKLNKLEHKIIPILSDV REVDVKGNRVIMNLPKFAHKFIDKALDIVEEGGVIHYYTIGKDFDKAIKLFEKKCDCEVLEKR IVKSYAPREYILALDFKINKK
Figure 3.7 DNA and protein sequence of codon-optimized TRM5. The DNA sequence incorporates NdeI and HindIII sites for cloning (underlined). The start and stop codons are shown in bold.
79 DNA sequence
CATATGATCCCGGAAGAAATCTATAAAATTCTGCGTAAACAGCGCTACCAAATTGATGGCCAT ACCGCAGTGAAACTGTGCGGTTGGGTTCGTAAGAAAATGCTGGAAGACAAAAACTGTTACAAA TCAAAATTCTACGGCATCGAAACCCACCGCTGCATTCAGTGTACGCCGTCGGTTATCTGGTGC CAGCAAAATTGCATTTTCTGTTGGCGTGTCCTGCCGCGCGATATTGGTATCGACATTTCACAA ATCAAAGAACCGAAATGGGAAGAACCGGAAGTGGTTTACGAAAAAATCCTGGCAATGCATAAA CGTATCATCATGGGCTACGCTGGTGTTCTGGATCGCGTCGGCGAGAAAAAATTTAAAGAAGCG CTGGAACCGAAACATGTGGCCATCAGTCTGTCCGGTGAACCGACCCTGTATCCGTACCTGGAT GAACTGATCAAAATTTTCCACAAAAACGGCTTTACCACGTTCGTCGTGAGCAATGGTATTCTG ACCGATGTTATCGAAAAAATTGAACCGACGCAGCTGTATATCAGTCTGGACGCGTATGATCTG GACTCCTACCGTCGCATTTGCGGCGGTAAAAAAGAATACTGGGAATCTATCCTGAACACCCTG GATATTCTGAAAGAGAAAAAACGTACGTGTATCCGCACCACGCTGATTCGTGGCTATAATGAT GACATCCTGAAATTTGTCGAACTGTACGAACGCGCTGATGTGCATTTCATTGAACTGAAAAGC TATATGCACGTGGGTTACTCTCAGAAACGTCTGAAAAAAGAAGATATGCTGCAACACGACGAA ATCCTGAAACTGGCGAAAATGCTGGATGAAAACAGCTCTTATAAACTGATTGATGACTCAGAA GACTCGCGTGTTGCCCTGCTGCAGAACGAAAATCGCAAAATTAATCCGAAACTGTAAAAGCTT Protein Sequence
MIPEEIYKILRKQRYQIDGHTAVKLCGWVRKKMLEDKNCYKSKFYGIETHRCIQCTPSVIWCQ QNCIFCWRVLPRDIGIDISQIKEPKWEEPEVVYEKILAMHKRIIMGYAGVLDRVGEKKFKEAL EPKHVAISLSGEPTLYPYLDELIKIFHKNGFTTFVVSNGILTDVIEKIEPTQLYISLDAYDLD SYRRICGGKKEYWESILNTLDILKEKKRTCIRTTLIRGYNDDILKFVELYERADVHFIELKSY MHVGYSQKRLKKEDMLQHDEILKLAKMLDENSSYKLIDDSEDSRVALLQNENRKINPKL Figure 3.8 DNA and protein sequence of codon-optimized TYW1. The DNA sequence incorporates NdeI and HindIII sites for cloning (underlined). The start and stop codons are shown in bold.
80 through Squire Booker, Pennsylvania State University, University Park, PA), which contains the isc operon of Azotobacter vinelandii145. One colony was grown overnight in 0.1 L of LB containing 34 µg/mL kanamycin and 100 µg/mL ampicillin at 37 °C. The overnight culture was distributed among six 2.8 L
Fernbach flasks containing 1 L LB, 100 µg/mL of ampicillin, and 34 µg/mL of kanamycin. The cells were grown to OD600 ~ 0.3 prior to induction of pDB1282 with arabinose (0.05 % w/v); TYW1 expression was induced by addition of 0.1 mM IPTG at OD600 ~ 0.5. Ferric chloride (50 µM) was also added at the time of induction by IPTG. Cells were harvested by centrifugation at 4,000 xg and frozen at – 80 °C.
Purification of TRM5
All purification steps were carried out at 4 °C. Cell paste (~ 12 g) was suspended in 20 mM Tris-HCl (pH 8.0) buffer containing 2 mM dithiothreitol
(DTT) and 1mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed using a
Branson digital sonifier at 50% amplitude and clear lysate was obtained by centrifugation at 18,500 xg. Since TRM5 is from a thermophile, we utilized a heat denaturation step to rapidly remove endogenous E. coli proteins by incubating the lysate at 80 °C for 30 min, followed by cooling in an ice bath for 15 min.
Precipitated proteins were removed by centrifugation at 18,500 xg. The lysate was brought to a concentration of 1.5 M (NH4)2SO4 by slow addition of an equal volume of 3 M (NH4)2SO4 while stirring on ice. The solution was loaded onto a butyl sepharose fast flow column (12.5 X 2.5 cm), which had been equilibrated in the
81 resuspension buffer containing 1.5 M (NH4)2SO4. The column was washed with 4 column volumes of loading buffer and the protein was eluted with a linear gradient to 0% (NH4)2SO4. The fractions containing TRM5 were identified by SDS-PAGE, pooled, and dialyzed against 4 L of 20 mM Tris-HCl (pH 8.0) buffer containing 50 mM KCl, and 2 mM DTT, with three buffer changes. The protein was concentrated initially with an Amicon pressure concentrator (YM-10 membrane), and then to the working concentration using an Amicon centrifugal concentrator (YM-10 membrane). Aliquots of the protein were frozen in liquid N2 stored at -80°C. A typical purification yielded approximately 20 mg/mL of protein that was greater than 95% pure as judged by SDS PAGE.
Purification of TYW1
TYW1 was purified in a Coy anaerobic chamber (98% N2, 2% H2). Cell paste (~10 g) was resuspended in 20 mM potassium phosphate (pH 7.2) buffer containing 0.5 M KCl, 50 mM imidazole, and 1 mM PMSF. Cells were lysed using a Branson digital sonifier at 50% amplitude at 4 °C. The cell lysate was cleared by centrifugation at 18,500 xg and a heat-denaturation step of endogenous proteins was carried out as described above for TRM5, except that the protein was removed from the chamber in a sealed glass container and returned to the chamber after the treatment for subsequent steps. A cleared lysate obtained by centrifugation at
18,500 xg at 4 °C was loaded onto a 1 mL HisTrapHP column (GE Healthcare) charged with NiSO4 and equilibrated in 20 mM potassium phosphate (pH 7.2), buffer containing 0.5 M KCl, and 50 mM imidazole as suggested by the
82 manufacturer’s protocols. The protein was eluted with a linear gradient from 0.05
– 0.7 M imidazole in 20 mM potassium phosphate (pH 7.2) buffer containing 0.5
M KCl. The brown fractions were pooled and desalted using an EconoPac 10DG disposable column (BioRad) that had been equilibrated in 0.05 M PIPES•NaOH
(pH 7.4) containing 2 mM DTT.
Reconstitution of TYW1
TYW1 eluted from the affinity column was treated with 8-fold molar excess of FeCl3 and Na2S at room temperature for 6 h. The resulting solution was centrifuged to remove precipitated components and desalted using an EconoPac
10DG column that had been equilibrated in 0.05 M PIPES•NaOH (pH 7.4) containing 2 mM DTT. Aliquots of the protein were concentrated and stored at -
80 °C. A typical purification yielded approximately 22 mg/mL of protein that was greater than 95% pure as judged by SDS PAGE.
Quantification of protein solutions
All protein stocks were quantified by Bradford assays with BSA as standard.
In vitro transcription of tRNA
A DNA oligonucleotide complementary to M. jannaschii phenylalanine tRNA gene (MJ-t16) with the T7 promoter at the 3’ end, and an oligonucleotide complementary to the T7 promoter were obtained from Eurofinns. The
83 oligonucleotides (10 µM each) were annealed in an Eppendorf thermocycler using the following conditions: 95°C for 20 seconds with a -0.5°C change in temperature each cycle for 34 cycles. The in vitro transcription was carried out using transcription buffer (Fermentas), 0.75 µM annealed oligonucleotides, 10 mM DTT,
10 mM MgCl2, 4 mM rNTPs (Fermentas), 1 U/mL inorganic pyrophosphatase, and
10% v/v recombinant T7 RNA polymerase (a gift Squire Booker). All reagents were RNase free. The reaction was incubated for 3 hours at 37 °C. DNase I was added at a volume of 1/25 the original reaction mixture and incubated at 37°C for
30 min. tRNA was extracted using miRNeasy column as previously described141.
Full-length transcript was separated from abortive transcripts on a HiPrep 16/60
Sephacryl S-200 gel filtration column (GE healthcare) developed isocratically with water. Fractions containing full length tRNA were pooled and concentrated using an Amicon centrifugal concentrator (YM-10 membrane).
Preparation of S-adenosyl-L-methionine (SAM)
SAM was synthesized using E. coli strain DM22-(pK8) (gift from George
Markham) as described previously146 except that the reaction mixtures contained acetonitrile (20 %v/v). After the reaction was complete, the pH was adjusted to 5 with HCl and the reactions were placed on ice for 15 minutes and centrifuged at
26,500 xg. The supernatant was diluted to 1 L with 1 mM sodium acetate (pH 5.0) and loaded onto a CM cellulose column (19.6 cm x 25 cm). The column was rinsed with 0.7 L of 1 mM sodium acetate and SAM was eluted with a gradient to 0.2 M
HCl over 0.9 L. Fractions containing SAM were identified by HPLC, pooled, and
84 lyophilized. The resulting residue containing SAM was resuspended with 4 mL
H2O and frozen in aliquots at –80 °C.
Protein assays
The assays were carried-out in an anaerobic chamber and RNase free, anaerobic solutions were used throughout. Experiments to determine the substrate for TYW1 were performed in 0.1 M Tris-HCl (pH 8.0) containing 4 mM DTT, 0.1 mM EDTA, 6 mM MgCl2, 100 mM KCl, and 2 mM SAM. The assay mixtures contained 50 µM TRM5, 50 µM TYW1, 10 mM sodium dithionite, 1.5 mM methyl viologen, and 24 µM M. jannaschii phenylalanine tRNA. Acetyl CoA, acetyl phosphate, phosphoenolpyruvate, or pyruvate was added to a final concentration of
1 mM. Reactions were incubated at 60 °C for 12 hours and were quenched by addition of 0.25 mL QIAzol reagent (Qiagen), which contains phenol and precipitates proteins. tRNA was extracted, digested to the nucleoside level and analyzed by LC-MS as described previously141.
85 Chapter 4:
Mechanistic studies of the radical S-adenosyl-L-methionine enzyme 4-
demethylwyosine synthase reveal the site of hydrogen atom abstraction
This work has been previously published and is reproduced with permission from:
Young, A. P., and Bandarian, V. (2015) Mechanistic studies of the radical S- adenosyl-L-methionine enzyme 4-demethylwyosine synthase reveal the site of hydrogen atom abstraction, Biochemistry 54, 3569-3572.
The manuscript was written by A. P. Young and V. Bandarian. All experiments were designed by A. P. Young and V. Bandarian. All experiments were performed by A. P. Young.
86 4.1 Abstract
TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m1G) with carbons 2 and 3 of pyruvate. In this study labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a deuterium labeled m1G methyl group was used to identify the methyl group of m1G as the site of hydrogen atom abstraction by 5¢-deoxyadenosyl radical.
13 2 [2- C1,3,3,3- H3]-Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.
4.2 Introduction
In the 58 years since the discovery of tRNA147 over one hundred modifications of the four canonical RNA bases have been identified that range in complexity from simple methylations of heteroatoms to the hypermodified bases queuosine and yW2.
yW and wyosine derivatives are modifications of a genetically encoded guanosine at position 37 of Phe encoding tRNA in eukaryotes and archaea9. The unique tricyclic core of imG-14 is both an intermediate in the biosynthesis of more complex wyosine derivatives in eukaryotes and archaea and a RNA base in archaea48. To date, eight structural homologs of imG-14 have been identified2.
The biosynthesis of imG-14 requires the successive actions of TRM5 and
TYW117,19,21, as shown in Figure 4.1. TRM5 is a class I SAM dependent methyl
87
Figure 4.1 Biosynthesis of imG-14
88 transferase that methylates N1 of guanosine 37 to produce m1G148,149. The key step involved in the formation of the tricyclic imG-14 core is catalyzed by TYW1, which converts m1G to imG-14 by adding two carbons that are derived from pyruvate, creating the imidazoline ring27. Additional species-specific modifications tailor the core wyosine base to form the known wyosine derivatives17,36,37,48.
TYW1 was identified as a member of the radical SAM superfamily on the basis of a characteristic CxxxCxxC motif22. The three Cys sidechains of this motif in radical SAM enzymes coordinate three irons of a cubane [4Fe-4S] cluster. SAM is coordinated to the fourth, or unique, iron via its amino and carboxylate groups83,150. Upon reduction of the +2 resting state of the cluster to the +1 state, the cluster reductively cleaves SAM forming the high energy dAdo• and methionine.
The dAdo• abstracts a hydrogen atom from a substrate to produce an intermediate that undergoes transformations culminating in products23. In some members of the radical SAM superfamily, the cofactor re-forms at the end of the catalytic cycle, whereas in others, SAM is used stoichiometrically151.
While two X-ray crystal structures of TYW1 are available, neither show electron density for the expected radical SAM [4Fe-4S] cluster24,25. In addition to the three Cys residues in the CxxxCxxC motif, three additional Cys residues are also found in the presumed active site clustered across from the radical SAM cluster binding site. These residues have been proposed to coordinate a second cluster.
Electron paramagnetic resonance studies have confirmed the presence of a second
[4Fe-4S] cluster26.
89 In a previous publication, we demonstrated that pyruvate is the source of two carbons in the imidazoline ring, the third being derived from the methyl of m1G27. However, significant gaps in our understanding of the mechanism of TYW1 remain. Herein we utilize isotopically labeled substrates to demonstrate that dAdo• is directly involved in hydrogen atom abstraction from the substrate and that the source of C6 and the C6 methyl of the imidazoline ring are derived from carbons 2 and 3 of pyruvate, respectively (see Figure 4.1 for numbering).
4.3 Results and discussion
To examine if dAdo•, resulting from the reductive cleavage of SAM, abstracts a hydrogen atom from m1G to initiate the catalytic cycle we synthesized tRNAPhe that contained either unlabeled or deuterated m1G and followed the transfer of hydrogen to dAdoH. The tRNAPhe substrate for these experiments, encoded by the M. jannaschii gene (MJ-t16), was produced by in vitro transcription and treated with
1 TRM5 and SAM to introduce the m G moiety. Unlabeled (CH3-SAM) or SAM containing a deuterated methyl group (CD3-SAM) were used to modify the tRNA producing the corresponding protiated or deuterated m1G. The synthetic tRNA substrates were incubated with TYW1 in the presence of pyruvate, and dAdoH produced under these conditions was analyzed by LC-MS. The ThermoFisher
Orbitrap XL mass spectrometer employed in these studies has mass accuracy of <5 ppm and sufficient resolution to differentiate isotopic content of dAdoH. Under the conditions of the experiment, dAdoH elutes at 43 min (Figure 4.2) and is readily assigned by comparison of retention time and mass spectra to the commercially
90 obtained dAdoH. The mass spectrum of dAdoH obtained from this analysis is shown in Figure 4.3. The peak at m/z 252.1087 corresponds to [M+H+] of dAdoH
(C10H14N5O3), and the measured mass is within 4 ppm of the theoretical value (m/z
252.1097). The isotope envelope at +1 m/z shows resolved peaks due to 15N, 13C, or D, which are present at 1.8, 10.8, and 0.1% of natural abundance, respectively, relative to the molecular ion. The black trace in Figure 4.3 shows the mass spectrum of dAdoH standard and shows peaks corresponding to the 15N isotope at m/z 253.1058, the 13C isotope at m/z 253.1141, and a barely discernible D isotope peak at m/z 253.1149. Therefore, the mass spectrum will readily allow the transfer of deuterium to dAdoH to be measured sensitively. The obvious advantage of the
Orbitrap detector over more traditional mass spectrometry instrumentation is the isotopic resolution, which allows for even small quantities of deuterium transfer to be detected.
The mass spectra of dAdoH produced when TYW1 was incubated in the presence of either protiated or deuterated tRNA and substoichiometric SAM are shown in Figure 4.3, with the traces normalized to the [M+H+] peak at m/z
252.1087. The red and blue traces correspond to the reaction performed with protiated or deuterated tRNA. At m/z 253.1149, there is a large peak present in the dAdoH produced in the presence of deuterated tRNA that is not present when protiated tRNA is used or in the standard. dAdoH containing one deuterium
(C10H13N5O3D) has an expected m/z 253.1159, which corresponds to a difference of 4 ppm to the 253.1149 obtained in the experiment. The dAdoH produced in the presence of deuterated tRNA has a 100-fold increase in the peak at m/z 253.1149
91
Figure 4.2 The extracted ion chromatogram at m/z 252 of dAdoH standard, and the dAdoH produced in the presence of protiated and deuterated substrate.
92
Figure 4.3 Mass spectrum of dAdoH produced in the presence of deuterated substrate (blue), protiated substrate (red), and standard dAdoH (black).
93 relative to that obtained with unlabeled SAM and that in the control sample. This peak corresponds to dAdoH containing a single deuterium. In the presence of deuterated m1G, the peak is 28% of the isotope peak. This data is consistent with dAdo• directly abstracting a hydrogen atom from the methyl group of m1G to initiate catalysis.
One would expect all of the dAdoH produced, in the presence of deuterated substrate to have a m/z of 253.1149 with no species at m/z 252.1097 corresponding to unlabeled dAdoH. The large background of protiated dAdoH is due to the abortive cleavage of SAM, wherein dAdo• abstracts a proton from a site other than the substrate. This phenomenon has been observed in nearly all radical SAM enzymes studied to date71. We opted to carry out the experiment with sub- stoichiometric SAM to reduce the background. There is no evidence for transfer of multiple deuteriums to dAdoH, as we see no peak at m/z 254.1222.
We next probed the fate of the remaining two protons on the methyl group of m1G. In these experiments, the modified tRNA produced in the reaction was digested enzymatically to nucleosides and analyzed by LC-MS. It was expected that one of the hydrogen atoms from the m1G methyl group would be retained in imG-14; surprisingly, however, the imG-14 produced with the labeled and unlabeled m1G gave rise to MS spectra with a peak at m/z 322.1137, as shown in
Figure 4.4, indicating that a deuterium was exchanged with a proton during the course of the experiment. To confirm that the starting substrate was fully deuterated, the mass spectra of the m1G digested to the nucleoside level from both the protiated and deuterated tRNA substrates were examined. The MS data clearly show that
94
Figure 4.4 Mass spectrum of imG-14 produced in the presence of protiated substrate in H2O (red), deuterated substrate in H2O (purple), protiated substrate in D2O (black), and deutertated substrate in D2O (blue). The peak due to deuterium incorporation is labeled. The natural abundance 13C peak is visable at m/z 323.1168.
95
Figure 4.5 Extracted ion chromatographs of digested protiated and deuterated tRNA showing that both samples show a peak with identical eleution time as commercial standard m1G, but at m/z of 298 with protiated tRNA and 301 with deuterated tRNA.
96
Figure 4.6 Mass spectra of m1G from the protiated or deuterated tRNA samples in Figure 4.5.
97 m1G is fully deuterated (see Figure 4.5-6), suggesting that the absence of m1G deuteriums is either mechanistically relevant, or occurs as part of the workup.
To determine if protons in imG-14 were exchanging with solvent, the reaction was repeated in D2O. Figure 4.4 shows the mass spectrum of imG-14 produced in both D2O and H2O with deuterated and protiated substrate but worked up in H2O. The predominant peak in all four cases is at m/z 322.1137, corresponding to product with no deuterium. Interestingly, when the reaction is performed with deuterated substrate in D2O, we observe 30-fold enrichment of a species with a single deuterium relative to that observed when protiated tRNA is turned over in
H2O. By contrast, when the reaction is performed in D2O using protiated tRNA, there is a 22-fold increase in the peak at 323.1196. These observations support the notion that one of the protons from the starting m1G is retained in imG-14, but there is significant exchange during workup of the reaction. Indeed, in a control experiment, in which the reactions were worked up in D2O, the predominant imG-
14 species has a peak at m/z 323.1194, which corresponds to C13H15N5O5D
(expected m/z 323.1215), indicating substantial proton exchange with solvent
(Figure 4.7). To pinpoint the site of exchange, we examined the mass spectrum of guanosine from the same LC-MS runs. The guanosine in the sample shows an elevated level of monodeuteration. However, the extent of exchange does not does not appear to be the same as with imG-14, as the predominant peak is that of unlabeled guanosine. Our interpretation of this result is that the substantial exchange observed in imG-14 occurs in the imidazoline ring, and not in the
98
Figure 4.7 The mass spectrum of imG-14 (panel A) and guanosine (panel B) produced when the digest is performed in D2O (red) and H2O (black).
99 guanine-like core of the molecule. Taken together with the data presented in Figure
4.4, our data suggest that one of the protons of m1G is retained in the final product.
Isotope labeling experiments have demonstrated that C2 and C3 of pyruvate are utilized to form the new carbons that are required to form the imidazoline ring of imG-14. However, these experiments did not provide insights into the regiochemistry of incorporation. To address this, we have conducted the TYW1-
13 2 13 catalyzed reaction with [2- C1, 3,3,3- H3]- and [2- C1]-pyruvate and analyzed the base produced in the incubation by LC-MS. As shown in Figure 4.4, unlabeled
13 imG-14 has a peak at m/z of 322. In the presence of [2- C1]-pyruvate, the mass of
13 2 the product shifts to m/z 323, whereas when [2- C1, 3,3,3- H3]-pyruvate is used, the mass shifts to m/z 326. Figure 4.8 shows the extracted ion chromatograms at
13 2 m/z 323 and 326 in the presence of both substrates. When [2- C1, 3,3,3- H3]- pyruvate is used as the substrate, the prominent peak is at m/z 326, which is consistent with conservation of all of the three deuteriums that are in the starting pyruvate. These data show that the methyl group of the imidazoline ring is formed from C3 of pyruvate and that C2 of pyruvate forms the bridging carbon, closing the imidazoline ring.
A possible mechanism for the transformation of m1G to imG-14 is shown in Figure 4.9. Our data clearly show that dAdo• abstracts a hydrogen atom from the methyl of m1G. The radical intermediate combines with pyruvate, which has variously been proposed to be activated by Schiff base formation to an absolutely conserved Lys27 or by interaction with a second [4Fe-4S] cluster, the presence of which has been inferred from spectroscopic measurements26. Oxidative or
100
Figure 4.8 Extracted ion chromatograms showing incorporation of deuterium into imG- 14 from pyruvate.
101
Figure 4.9 A proposed mechanism for the formation of imG-14.
102 reductive cleavage of the intermediate and transimination lead to an intermediate, which eliminates the electrophilic center to form products.
In summary, this study has identified that hydrogen atom abstraction by dAdo• occurs at the methyl of m1G and established the regiochemistry of the pyruvate incorporation. The role of the iron-sulfur clusters and the fate of C1 of pyruvate remain to be determined.
4.4 Materials and methods
Cloning of TRM5 and TYW1
The codon optimized gene, obtained from GenScript, corresponding to
TRM5 (MJ_0883) was excised from pUC57 and cloned into the NdeI and HindIII sites of pET28a. The cloning of TYW1 was described previously in Chapter 3.427.
Expression of TRM5 and TYW1
Both proteins were expressed as described previously in Chapter 3.427.
Purification of TRM5
All purification steps were carried out at 4 °C. Cell paste (~20 g) was suspended in 0.1 L of 20 mM tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer at pH 8 containing 2 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysis was performed using a Branson sonifier at 50 % amplitude with 24x 30 sec bursts, with 1 min between each cycle to maintain the solution near 4 °C. Clarified lysate was obtained by centrifugation
103 at 15,000 x g at 4°C, then heated to 80 °C for 30 min, and then cooled rapidly in an ice bath. Precipitated proteins were removed by centrifugation at 15,000 x g at 4°C and loaded onto a 40 mL DEAE Sepharose fast flow column equilibrated in 20 mM
Tris-HCl buffer at pH 8 containing 2 mM DTT. The column was washed with 4 column volumes (160 mL) of loading buffer and bound protein was eluted with a gradient to 1 M sodium chloride (NaCl) in the starting buffer over 0.4 L. Fractions containing TRM5 were identified by SDS-PAGE and were pooled. TRM5 was further purified by loading the pooled fractions onto a 5 mL HiTrap chealting HP column charged with nickel chloride (NiCl2) and equilibrated with 50 mM potassium phosphate (KPi) buffer at pH 7.4 containing 0.5 M potassium chloride
(KCl), and 50 mM imidazole. The column was washed with the loading buffer and bound protein was eluted with a gradient to 0.5 M imidazole in the starting buffer over 50 mL. Fractions containing TRM5 were identified by SDS-PAGE, pooled, and concentrated using an Amicon concentrator (YM-10 membrane). The concentrated protein was desalted over a Sephacryl 16/60 S-200 column that had been equilibrated in 20 mM Tris-HCl (pH 8.0) buffer containing 0.25 M sodium chloride, and 2 mM DTT. The peak corresponding to monomeric protein was pooled, concentrated and aliquots were flash frozen in liquid nitrogen.
Purification of TYW1
TYW1 was purified in a Coy anaerobic chamber (95 % N2, 5 % H2-98 %
N2, 2 % H2) at room temperature. Cell paste (30 g) was suspended in 0.2 L of 50 mM KPi buffer at pH 7.4 containing 0.5 M KCl, 50 mM imidazole, and 1 mM
104 PMSF. Cells were lysed using a Branson sonifier at 50 % amplitude with 24x 30 sec bursts with 1 min in between each cycle to keep the solution at 4 °C. The lysate was clarified by centrifugation at 15,000 x g at 4°C and loaded onto two 5 mL
HiTrap chealting HP columns that had been serially connected, charged with NiCl2, and equilibrated with 50 mM KPi buffer at pH 7.4 containing 0.5 M KCl, and 50 mM imidazole. Bound protein was eluted by washing with the loading buffer containing 0.5 M imidazole. Colored protein fractions were pooled and brought to
~ 1 M ammonium sulfate by the addition of solid ammonium sulfate while stirring on ice. The protein was loaded onto a 40 mL butyl Sepharose Fast flow column pre-equilibrated with 20 mM Tris-HCl buffer at pH 8 containing 1 M ammonium sulfate. Adsorbed protein was eluted by a step gradient to 20 mM Tris-HCl buffer at pH 8. Brown fractions were pooled, concentrated using an Amicon concentrator
(YM-10 membrane), and desalted into 50 mM PIPES-NaOH buffer at pH 7.4 containing 10 mM DTT using a EconoPac 10DG disposable column.
Reconstitution of the iron sulfur clusters was performed with desalted
TYW1 by stirring at room temperature with a 10-fold molar excess of iron(III)chloride and sodium sulfide for 4 h. Precipitated protein was removed by centrifugation and the resulting sample was exchanged into 50 mM PIPES-NaOH buffer at pH 7.4 containing 0.15 M KCl, and 2 mM DTT. Desalted protein was concentrated using an Amicon (YM-10 membrane) and loaded onto a Sephacryl
16/60 S-200 column equilibrated in the same buffer as the protein. The peak corresponding to the monomeric protein was pooled, concentrated, and flash frozen in liquid nitrogen.
105
Determination of protein concentrations
The concentration of TRM5 and TYW1 were determined using Bradford assay with BSA as standard. A Bradford correction factor of 0.32 for TYW1, which was determined for TYW1 as previously described74, was applied.
Preparation of S-adenosyl-L-methionine (SAM)
SAM containing a protiated methyl group was prepared as previously described in Chapter 3.427. SAM containing a deuterated methyl group was prepared using the same method as above except that L-methionine (methyl-D3) replaced unlabeled L-methionine. The positive ion ESI-MS of protiated and deuterated SAM are shown in Figure 4.10. The deuterated sample is at least 98% deuterated, as judged by comparison of the peaks at m/z of 399 and 402.
Purification of Saccharomyces cerevisiae tRNA. S. cerevisiae
YPL207W knockout strain (YSC1021-547084 Thermo Scientific) was grown in yeast, peptone, dextrose media (1% yeast extract, 2% peptone, and 2% dextrose) media at 30 °C and shaking at 200 rpm to OD600 ~ 1. Cells were harvested by centrifugation at 5,000 x g at 4 °C for 10 minutes, flash frozen in liquid nitrogen, and stored at -80 °C.
106
Figure 4.10 The mass spectrum of S-adenosyl-[methyl-D3]-methionine.
107 Soluble RNA was extracted from yeast cells using the following published methods26,152 as follows. Cells were resuspended in an equal ratio (w/v) of 10 mM
Tris-HCl (pH 8.0) buffer containing 10 mM MgCl2 and 0.15 M NaCl. An equal volume of liquified phenol (pH 4.3) (Fisher) was added and the suspension was stirred at room temperature for 1 h. Cell debris was removed by centrifugation at
1,500 x g for 10 min at room temperature. An equal volume of liquified phenol (pH
4.3) was added to the supernatant, the solution was mixed, and centrifuged at 7000 x g for 20 minutes at 4°C. The supernatant was removed and soluble RNA was precipitated by addition of 0.1 volume of 3 M sodium acetate pH 5.2, and 3 volumes of ethanol. The mixture was placed at -80°C overnight. RNA was pelleted by centrifugation at 15,000 x g for 30 min at 4°C. The resulting pellet was resuspended in ice cold 1 M NaCl and centrifuged at 7,000 x g at 4°C for 30 minutes. The supernatant was removed, the pellet was resuspended in ice cold 1 M NaCl, and centrifuged again. The supernatant was pooled with that from the previous step and the RNA was precipitated with sodium acetate and ethanol as described above. The resulting pellet was resuspended in RNAse free water and the concentration was
-1 -1 determined by A260 (ε= 0.025 (µg/mL) cm ).
Methylation of tRNA
M. jannaschii phenylalanine tRNA was produced by in vitro transcription as previously described, as described in Chapter 3.427. TRM5 was used to methylate the tRNA under the following assay conditions: 0.1 M Tris-HCl (pH 8.0), 0.1 M
KCl, 4 mM DTT, 2 mM MgCl2, 2 mM SAM, 16 µM TRM5, and 184 µM tRNA
108 produced via in vitro transcription. To produce protiated tRNA SAM-[methyl-H3] was used and to produce deuterated tRNA SAM-[methyl- D3] was used. Reactions were incubated at 50°C for 4 h and were quenched by addition of Qiazol (Qiagen). tRNA was extracted as previously described5 and concentration was determined by
-1 -1 A260 (ε= 0.025 (µg/mL) cm ).
Enzyme assays
The assays to determine the site of hydrogen atom abstraction were performed in
0.1 M Tris-HCl (pH 8.0) buffer containing 0.1 M KCl, 4 mM DTT, 75 µM SAM,
2 mM pyruvate, 10 mM sodium dithionite, 1.5 mM methyl violagen, 0.1 mM methylated tRNA, and 0.1 mM TYW1. Reactions were incubated at 50°C for 30 min. At the end of the incubation, the samples were split into two and one half was analyzed for deuterium incorporation into dAdoH, and the other half for the RNA modification. dAdoH production was assayed by filtering the samples through a centrifugal concentrator with a 10,000 molecular weight cut off. An aliquot (40 µL) of the filtrate was analyzed by HPLC, which had been interfaced to a LTQ Orbitrap
XL mass spectrometer essentially as described141 except that the 50 mM ammonium actetate (pH 6.0) replaced the 1.93% (w/v) ammonium acetate (pH 6.0) used previously. The sample that was analyzed for RNA modification was quenched by the addition of 0.25 mL Qiazol and 50 µL chloroform. The RNA was extracted and digested to the nucleoside level as previously described141.
The assays performed in D2O were identical to the one described above with the following exceptions. The KCl, DTT, pyruvate, sodium dithionite, and methyl
109 violagen were all resuspended in D2O. The Tris-DCl (pH 8.4) was prepared by dissolving the Tris base in D2O and adding DCl until the pH was 8.4. TYW1, SAM, and tRNA were all exchanged into D2O by lyophilizing and resuspending in an equal volume of D2O.
The digestion of tRNA in D2O was performed as follows. The enzyme assay was performed in 0.1 M Tris-HCl (pH 8.0) buffer containing 0.1 M KCl, 4 mM
DTT, 75 µM SAM, 2 mM pyruvate, 10 mM sodium dithionite, 1.5 mM methyl violagen, 200 µg yeast tRNA, and 0.1 mM TYW1. Reactions were incubated at
50°C for 30 min. tRNA was extracted as described above with the exception that the sample that was to be analyzed in D2O was eluted with 50 µL of D2O. The buffers that were used to perform the digestion in D2O were prepared as follows.
The 0.1 M ammonium acetate (pH 5.3) was prepared by dissolving ammonium acetate in D2O and adjusting the pH with DCl. 1 M ammonium bicarbonate was prepared by dissolving the salt in D2O. Finally, the 10 mM Tris-HCl (pH 7.5) buffer containing 10 mM MgCl2 was performed by dissolving Tris base and MgCl2 in D2O and adjusting the pH with DCl. The digestion was performed in both D2O and H2O and analyzed via LC-MS as previously described141.
The assays to determine the regiochemistry of the pyruvate derived carbons were performed in 0.1 M Tris-HCl (pH 8.0) buffer containing 0.1 M KCl, 4 mM
13 2 13 DTT, 2 mM SAM, 50 µM TYW1, 1 mM [2- C1 3,3,3- H3] or [2- C1] pyruvate,
10 mM sodium dithionite, and 200 µg yeast tRNA. The reactions were allowed to proceed overnight at 50°C. The RNA was extracted, digested to nucleosides and analyzed via LC-MS as previously described27.
110 Chapter 5:
Biochemical characterization of Schiff base intermediate that forms during
4-demethylwyosine biosynthesis
All experiments were designed by A. P. Young and V. Bandarian. All experiments were performed by A. P. Young.
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5.1 Abstract
The radical SAM enzyme TYW1 catalyzes the condensation of pyruvate and m1G containing tRNAPhe, resulting in the formation of imG-14. During the course of the reaction a Schiff base is hypothesized to form between a conserved lysine residue in the protein and carbon two of pyruvate forming an electrophilic center that is subsequently attacked by the amine on carbon two of m1G forming the tricyclic ring that is characteristic of wyosine derivatives. This chapter describes site directed mutagenesis, of two conserved residues in TYW1, as well as detection and identification of a Schiff base between pyruvate and the amino group of the side chain of Lys41 in the M. jannaschii TYW1 homolog.
5.2 Introduction
There are over one hundred and fifty documented modified RNA bases found throughout the three domains of life1,2. The majority, over one hundred, of them are found in tRNA and range greatly in both complexity and function1,2.
Position 37, the position adjacent to the anticodon, usually contains a modified base. In tRNAPhe this base is almost always a hypermodified purine, with a modified adenine residue present in bacteria and a hypermodified guanosine residue known as a wyosine derivative present in both archaea and eukarya6,17,48. The presence of a large bulky base at this position has been shown to be important in preventing -1 frameshifting61,65.
All wyosine derivatives are modifications of the genetically encoded guanosine found at position 37 of phenylalanine encoding tRNA18. Guanosine is
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initially methylated at N1, forming m1G, by the SAM dependent methyltransferase
TRM519,20. The next step is formation of imG-14, the first base to contain the tricyclic base that is characteristic of all wyosine derivatives by the radical SAM enzyme TYW117,21. TYW1 catalyzes the condensation of carbons two and three of pyruvate and m1G with carbon one of pyruvate lost as a yet unidentified second product, as shown in Figure 5.126,27,36,37,153. Subsequent tailoring reactions form a wide variety of wyosine derivatives in both archaea and eukarya17,42,45,48.
The radical SAM superfamily consists of more than 100,000 members66 and is characterized by the presence of a CxxxCxxC motif22. Subsequently it has been shown that the CxxxCxxC motif may contain additional residues between the cysteines, but in all cases the three cysteine residues coordinate a 4Fe-4S cluster67,75. Each cysteine residue coordinates an iron, leaving a remaining uncoordinated, so-called, ‘unique’ iron. The unique iron has been shown by crystallography28,73,75,83, EPR26,30,32, Mössbauer spectroscopy26,31, and selenium X- ray absorption spectroscopy34,35 to ligate a molecule of SAM via by the carboxylate and amino moieties of the methionine portion. The resting state of the 4Fe-4S cluster is the +2 state and upon reduction it enters the catalytically active +1 state.
In the catalytically active state, inner sphere electron transfers from the cluster to
SAM causes homolytic cleavage of the sulfonium 5′-ribose bond forming methionine and the highly reactive intermediate dAdo•, which goes on to abstract a hydrogen atom from a substrate, forming a substrate radical and dAdoH. The
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Figure 5.1 The biosynthetic pathway for wybutosine from guanosine showing the reaction catalyzed by TYW1.
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substrate radical then goes on to form product, which may involve radical transfer to another molecule23. Members of the radical SAM family catalyze a wide variety of reactions which range from DNA repair70, cofactor biosynthesis67, RNA modification17, and small molecule biosynthesis154.
An EPR and Mössbauer study revealed the presence of a second 4Fe-4S cluster26, which had been hypothesized to exist based upon the clustering of three cysteine residues in the secondary structure of two crystal structures of TYW1 from
M. jannaschii25 and P. horikoshii24. This study proposed that pyruvate interacts with the unique iron of the second 4Fe-4S cluster, coordinating pyruvate in the active site26. The interaction of substrate with the second Fe-S cluster of a radical
SAM enzyme has precedent in the case of MoaA where GTP has been shown to interact with the second cluster in both a crystal structure28 and through an EPR study104. The observation of the interaction between pyruvate and the cluster led the authors to propose a mechanism that involved coordination of pyruvate to the second 4Fe-4S cluster26.
There are two proposed mechanisms for the transformation of m1G into imG-14 by TYW1 in the literature26,27. Both mechanisms share an initial hydrogen atom abstraction by dAdo• at the m1G methyl group, which was shown to occur in the work presented in Chapter 426,27,153. The mechanisms differ in how pyruvate is activated initially. The mechanism proposed by Young et al. uses a pyruvate Schiff base intermediate formed between pyruvate C2 and an absolutely conserved lysine residue, that has been shown to be required for activity in an in vivo complementation assay25, to activate the pyruvate and immobilize it in the active
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site27. The active site of TYW1 P. horikoshii homolog is shown in Figure 5.2 (all numbering of residues are that of M. jannaschii), there are two sets of conserved cysteine residues that are hypothesized to coordinate the two clusters on opposite sides of the active site channel. The cysteines proposed to ligate the radical SAM cluster are; C62, C66, and C69; while those that are proposed to ligate the auxiliary cluster are C26, C39, and C52. The lysine that is hypothesized to form a Schiff base is K41 and is shown close to the auxiliary cluster binding cysteines. The mechanism proposed by Perche-Letuvée et al. uses the second 4Fe-4S cluster to coordinate pyruvate in the active site, along with homolytic cleavage of the C1-C2 pyruvate bond forming an m1G pyruvate adduct. The keto oxygen of C2 of pyruvate is then lost as water, facilitated via it’s interaction with the cluster26. The two mechanisms are shown in Figure 5.3.
Schiff bases have been proposed to form in a number of different enzymes, such as aldolases155,156, rhodopsins157, decarboxylases158, and PLP159 dependent enzymes. Schiff bases have been characterized that form between both lysine residues within a protein155,156,159-161 and the amino terminus162,163. Schiff base intermediates are typically detected via borohydride or cyanoborohydride reduction and site directed mutagenesis. This chapter describes trapping and characterization of a Schiff base between TYW1 and pyruvate by sodium cyanoborohydride
(NaCNBH3).
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Figure 5.2 The active site of P. horikoshii TYW1 (numbering used is that of M. jannaschii). The three cysteines on the left; C62, C66, and C69; are hypothesized to coordinate the radical SAM 4Fe-4S cluster. On the other side of the channel are the three cysteines hypothesized to coordinate the auxiliary 4Fe-4S cluster. Located close to those cysteines is K41, the lysine residue proposed to form the Schiff base with pyruvate.
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Figure 5.3 The two proposed mechanisms for TYW1. Panel A shows the mechanism proposed by Young et al. that contains the Schiff base and panel B shows the mechanism proposed by Perche-Letuvée et al.
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5.3 Results and discussion
A C195S mutant of M. jannaschii TYW1 was created because a preliminary
X-ray structure (solved by Drennan lab in collaboration) revealed the presence of a
2Fe-2S cluster in place of the anticipated 4Fe-4S radical SAM cluster. This structure contained a disulfide between C195 and one of the cluster cysteines. This mutant was created to remove the possibility of this disulfide forming and preventing the cluster from forming.
Previous studies have demonstrated that M. jannaschii TYW1 can utilize bulk S. cerevisiae RNA as a substrate26,153. Figure 5.4 shows the extracted ion chromatogram at m/z 322 when bulk S. cerevisiae tRNA is digested to the nucleoside level following an incubation with wild type TYW1, C195S-TYW1 variant, and no TYW1. There is a peak present in the wild type TYW1 and C195S
TYW1 samples that has a m/z of 322, [imG-14+H+], with a retention time of 41.5 minutes that isn’t present in the absence of TYW1. The presence of this peak in both the wild type sample and mutant sample indicates that C195S-TYW1 variant is active.
Figure 5.5 shows the extracted ion chromatogram at m/z 322 of S. cerevisiae bulk tRNA that was digested to the nucleoside level following an incubation in the presence of wildtype TYW1, or the K41A variant, or no TYW1. A peak with m/z
322 was present only in the presence of the wild type TYW1 protein, indicating that the K41A mutant is inactive which is consistent with the results of a prior in vivo complementation assay25. Were a Schiff base forming between K41 of M.
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Figure 5.4 The extracted ion chromatogram at m/z 322 in the presence of wild type TYW1, C195S-TYW1, or no TYW1.
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Figure 5.5 The extracted ion chromatogram at m/z 322 in the presence of wild type TYW1, K41A-TYW1, or no TYW1.
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jannaschii TYW1 as proposed in the mechanism, shown in Figure 5.3, K41 would be required for activity.
To unambiguously demonstrate Schiff base formation between TYW1 and pyruvate; experiments were carried out where TYW1 was incubated with pyruvate, treated with NaCNBH3 and iodoacetamide, and the corresponding fragments obtained following a trypsin digest were analyzed by LC-MS. The iodoacetamide treatment was to ensure that the Cys sidechains are not oxidized in the course of the workup complicating the mass spectral analysis. Theoretically, the smallest fragment of M. jannaschii TYW1-C195S containing K41 that would result from a trypsin digest would be the peptide NCYK (the blue residue indicates modified cysteine and the red the modified lysine) assuming no missed cleavages by trypsin, and NCYKSK with one missed cleavage. A NCYK fragment, treated with iodoacetamide, would have an expected m/z 584.2497 [M+H+] and the peptide pyruvate adduct would have a m/z 656.2708 [M+H+]. If trypsin were to not cleave at K41, which is entirely possible as that residue may be modified with pyruvate and therefore protected from trypsin cleavage, the resulting peptide would be
NCYKSK. This NCYKSK fragment treated with iodoacetamide would have an expected m/z 799.3767 [M+H+] and its pyruvate adduct would have a m/z 871.3978
[M+H+]. The structures of the peptides and the corresponding modifications are shown in Figure 5.6. To unambiguously establish that pyruvate was the source of
13 13 the modification [1- C1]- and [1,2,3- C3]-pyruvate were employed as the source of the pyruvate in these experiments.
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Figure 5.6 The structures of the possible peptides resulting from a trypsin digest of M. jannaschii TYW1. The red residue is the hypothesized site of modification and the adduct is shown in red. The blue is the carbamidomethyl resulting from iodoacetamide treatment.
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Figure 5.7 shows the TIC and extracted ion chromatograms at m/z 871 and 874 of
13 TYW1 that has been incubated with no pyruvate and 1,2,3- C3-pyruvate, followed
by modification by iodoacetamide and NaCNBH3 (or sodium cyanoborodeuteride
(NaCNBD3)), and digestion with trypsin. The peptide corresponding to
NCKYKSK elutes at 6.3 min. Figure 5.8 shows the MS spectra from 6.2 to 6.8 min. When TYW1 is incubated with NaCNBH3 in the absence of pyruvate there is a peak present with a m/z 871.3993 (Figure 5.8A), which is consistent with peptide containing a carbamidomethyl modification and pyruvate adduct. This peak does not change (m/z 871.3986) when the reaction is quenched with NaCNBD4 (Figure
5.8B), consistent with the absence of a shift in the envelope.
The peak at m/z 871.3993 is 1.7 ppm from the theoretical m/z of a NCYKSK containing both the carbamidomethyl modification and the pyruvate adduct. This peak is present in the control containing no pyruvate and does not shift with either
NaCNBH3 or NaCNBD3. One possible explanation for this is that the protein binds pyruvate within the cell, forming a Schiff base. The resulting complex is reduced, either within the cell or during purification, forming a covalent adduct with the protein, rendering it inactive. In order to differentiate between protein that purified with the adduct bound and adduct formed during the experiment, all of the pyruvate used in the experiment contained 13C labels.
13 In contrast, when TYW1 is incubated with [1- C1]-pyruvate in the presence of NaCNBH3 there is a peak with a m/z 871.3993, which was detected in the sample containing no pyruvate, and a new peak with a m/z 872.4025 (Figure 5.8C). The theoretical m/z of the peptide pyruvate adduct containing one 13C is 872.4018
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Figure 5.7 The total ion count and extracted ion chromatograms at m/z 871 and 874 for trypsin digested TYW1 treated with NaCNBH3 in either the absence of 13 pyruvate or presence of [1,2,3- C3]-pyruvate.
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Figure 5.8 The mass spectra of trypsin digested TYW1 showing the NCYKSK peptide obtained when TYW1 was incubated with no pyruvate (spectra A & B), 13 13 1- C1-pyruvate (spectra C & D), and 1,2,3- C3-pyruvate (spectra E & F) in the presence of NaCNBH3 (spectra A, C, & E) and NaCNBD3 (spectra B, D & F).
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[M+H+], which is 1.5 ppm different to the experimentally obtained m/z. The
872.4025 peak is also present in the two samples without added pyruvate, due to the naturally occurring 13C isotopes found in the peptide. However, based upon the natural abundance of 13C, the 872.4018 peak should be 32% of the 871.3993 peak
13 theoretically, which it is in Figure 5.8A,B,C, however when [1- C1]-pyruvate is used and NaCNBH3 is used to reduce the adduct; the 872.4018 peak is significantly larger than that at 871.3993, indicating that this is due to the labeled pyruvate and not just +1 peak of the 871.3993 peak. When this experiment is performed in the presence of NaCNBD3, in addition to the peak at m/z 871, there is a peak at m/z
873.4084 (Figure 5.8D). A peptide pyruvate adduct containing one 13C and one deuterium has a theoretical m/z 873.4081 [M+H+], which is within error of the
13 experimentally determined m/z. When TYW1 is incubated with [1,2,3- C3]- pyruvate and NaCNBH3 we observe a new peak at m/z 874.4085, which is identical to the theoretically determined mass of the peptide pyruvate adduct containing three
13 + C is 874.4085 [M+H ]. When NaCNBD3 replaces NaCNBH3 a new peak with m/z 875.4156 appears (Figure 5.8F), which is within 1 ppm of the theoretical m/z of the peptide pyruvate adduct containing three 13C and one deuterium is 875.4148
[M+H+]. This data collectively establishes that TYW1 can be modified by pyruvate at a Lys residue within the peptide NCYKSK. At this point it is not possible to differentiate the site of modification, as there are two Lys residues present in the peptide. However, we suspect based on the proximity of the Lys residue to the conserved Cys residues in the X-ray crystal structure, sequence conservation, and site directed mutagenesis experiments that K41 is the target of modification.
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In control experiments the reactions were repeated in the presence or absence of various components to determine which components were required for the
13 modification (Figure 5.9). In these experiments [1,2,3- C3]-pyruvate was used to help differentiate the newly introduced adduct from the one that purifies with the
protein. Moreover, the spectra of samples treated with NaCNBH3 and NaCNBD3 were examined to show that in each experiment, the peak arose from reduction of a Schiff base. The data show that the formation of the Schiff base is independent of SAM, dithionite and tRNA. However, it is not observed when the NaCNBH3 or NaCNBD3 are omitted, presumably because it undergoes hydrolysis during the workup.
The radical SAM enzyme TYW1 catalyzes the condensation of m1G containing tRNA with pyruvate, forming imG-14 containing tRNA27.
Isotopologues of pyruvate showed that carbons two and three are incorporated into imG-14 with carbon three forming the methyl group and carbon two the bridging carbon in the five membered imiadozoline ring153. Using m1G containing tRNAPhe labeled at the methyl group with deuterium, hydrogen atom abstraction was shown to occur at the methyl group153. Two mechanisms have been proposed for this transformation, one of them containing a Schiff base between K41 and carbon two of pyruvate27. This will activate pyruvate for subsequent catalysis. The other proposed mechanism utilizes the cluster for activation of pyruvate26, and that group has proposed a role for the lysine in regulating the redox properties of the auxiliary cluster37.
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Figure 5.9 The mass spectra of trypsin digested TYW1 showing the NCYKSK peptide obtained when TYW1 was incubated with no SAM (spectra A & B), dithionite (spectra C & D), tRNA (E & F), TYW1 (G & H), and NaCNBH3 (spectrum I); in the presence of NaCNBH3 (spectra A, C, E, & G) or NaCNBD3 (spectra B, D, F, & H).
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If a Schiff base were to form between K41 of M. jannaschii TYW1 as proposed in the mechanism, shown in Figure 5.3, K41 would be required for activity. Figure 5.5 shows that the K41A variant is inactive. The alanine mutant being inactive is consistent with the Schiff base hypothesis as an alanine residue within a protein does not contain the free amine required for formation of a Schiff base, but this may not be the reason for the protein’s inactivity. For example, the
K41A mutant may be inactive as the proteins structure has changed causing substrate (which could be tRNA, SAM, or pyruvate in this case) not to bind.
Another hypothesis that has been proposed for the role of this lysine in the reaction is that it plays a role in controlling the redox state of the second 4Fe-4S cluster37, which would also account for the inactivity of the K41A mutant, both in vitro as shown above and in vivo25. In order to provide more evidence for the presence of a
Schiff base at K41, chemical trapping of the intermediate was required.
Schiff base enzyme intermediates have been trapped in the past using both
164-166 NaCNBH3 and NaBH4 . Figure 5.4 and 5.5 show that when TYW1 is incubated with both pyruvate and NaCNBH3 or NaCNBD3, treated with iodoacetamide, and trypsin digested, a fragment with m/z consistent with a peptide containing both a carbamidomethyl and reduced pyruvate adduct. This peptide fragment contains two lysine residues, K41 and K43, so it is impossible to say definitively that the Schiff base forms on residue K41. In order to definitively show the location of the adduct,
MS/MS fragmentation and subsequent sequencing of the peptide is required.
The data shown in Figure 5.9 shows that the two other substrates of TYW1,
SAM and tRNA, are not required for Schiff base formation between pyruvate and
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TYW1, suggesting that pyruvate can either bind first or that the binding order of the substrates is unimportant. This data also showed that the presence of dithionite is not required for Schiff base formation, which means that the oxidation states of the clusters do not affect binding of pyruvate to the substrate.
This chapter has demonstrated that the K41A mutant of M. jannaschii
TYW1 is required for activity, confirming the previously published in vivo
25 complementation results . With the use of NaCNBH3 a Schiff base was trapped between residues 38 and 43 of M. jannaschii TYW1 and pyruvate, it could be located on either K41 or K43. In order to determine which residue the Schiff base is trapped at fragmentation of the peptide and subsequent sequencing of the ions is required. The mass of the trapped covalent intermediate was shown to shift depending upon the pyruvate used in the experiment. In order to show definitively that the Schiff base is a true intermediate in the catalytic cycle chemical rescue of the K41A mutant is required using a primary amine such as methylamine as the lysine surrogate. The data provides support for the mechanism proposed by Young et al in which TYW1 forms a Schiff base with pyruvate during the course of the reaction27.
5.4 Materials and methods
Cloning of TYW1
The codon optimized gene corresponding to TYW1 (MJ_0257), obtained from Genscript with the sequence shown in Figure 3.8, was cloned into pET28t167 using the NdeI and HindIII cut sites as described in Chapter 327.
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Construction of C195S-TYW1 and K41A-TYW1 variant
The C195S-TYW1 variant was constructed using the Stratagene QuickChange site- directed mutagenesis protocol, with the primers shown in Table 6.1, and TYW1 pET28t construct as a template. The K41A-TYW1 variant was constructed using the Stratagene QuickChange site-directed mutagenesis protocol, with the primers shown in Table 5.1, and TYW1 pET28a construct as a template.
Expression of TYW1 variants
TYW1, both wild type and variants, were grown and expressed as previously described in Chapter 3 for TYW1 in pET28a27.
Purification of TYW1 variants
TYW1 was purified in a Coy anaerobic chamber (97.5% N2, 2.5% H2) at room temperature. Cell paste was resuspended in 50 mM potassium phosphate (pH
7.4) buffer containing 0.5 M potassium chloride, 50 mM imidazole, and 1 mM
PMSF. Cell paste was sonicated on ice with stirring for 12 minutes with 3 seconds on and 6 seconds off using a Branson sonifier at 50% amplitude. The lysate was clarified by centrifugation at 18,500 x g at 4°C for 30 minutes. The clarified lysate was then placed in an 80°C water bath for 30 minutes, followed by cooling in an
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TYW1 Primer Sequence variant K41A 5’-CAAAAACTGTTACGCATCAAAATTCTAC 5’-GTAGAATTTTGATGCGTAACAGTTTTTG C195S 5’-CCTACCGTCGCATTTCCGGCGGTAAAAAAGAATAC 5’-GTATTCTTTTTTACCGCCGGAAATGCGACGGTAGG
Table 5.1 The primers used to make the C195S and K41A variants of M. jannaschii TYW1. The bases mutated to create the variants are shown in bold.
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ice bath for 15 minutes. The lysate was again clarified by centrifugation at 18,500 x g at 4°C for 30 minutes. The clarified lysate was loaded onto a 5 mL HiTrap chealting HP column charged with nickel sulfate and equilibrated in 50 mM potassium phosphate (pH 7.4) buffer containing 0.5 M potassium chloride, and 50 mM imidazole. Bound protein was eluted with a step gradient to 50 mM potassium phosphate (pH 7.4) buffer containing 0.5 M potassium chloride, and 0.5 M imidazole. Solid ammonium sulfate was added to the pooled brown fractions to a final concentration of 1 M, after which it was loaded onto a 5 mL HiTrap Butyl HP column equilibrated in 20 mM Tris-HCl (pH 8.0) buffer containing 1 M ammonium sulfate. Bound protein was eluted via a step gradient to 20 mM Tris-HCl (pH 8.0).
Brown fractions were pooled, and desalted into 50 mM PIPES-NaOH (pH 7.4) buffer containing 2 mM DTT using a EconoPac 10DG disposable column.
The His tag was cleaved by an overnight incubation in the presence of TEV protease at a molar ratio of 20 mols of TYW1 to 1 mol of TEV protease, which was purified as previously described167. The following day the solution was loaded onto a 5 mL HiTrap chealting HP column charged with nickel sulfate and equilibrated in 50 mM potassium phosphate (pH 7.4) buffer containing 0.5 M potassium chloride, and 50 mM imidazole. The column was washed with equilibrating buffer and brown fractions were pooled and desalted into 50 mM PIPES-NaOH (pH 7.4) buffer containing 10 mM DTT using a EconoPac 10DG disposable column.
Reconstitution of the iron sulfur clusters was performed by stirring TYW1 at room temperature for 4 hours with a 10-fold molar excess of iron(III)chloride and sodium sulfide. After 4 hours precipitated material was removed by
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centrifugation and TYW1 was desalted into 50 mM PIPES-NaOH (pH 7.4) buffer containing 150 mM potassium chloride, and 2 mM DTT using a EconoPac 10DG disposable column. Desalted protein was concentrated to approximately 1 mL using an Amicon (YM-10 membrane) and loaded onto a Sephacryl 16/60 S-200 column equilibrated in 50 mM PIPES-NaOH (pH 7.4) buffer containing 150 mM potassium chloride, and 2 mM DTT. The peak corresponding to monomeric TYW1 was pooled, concentrated, and flash frozen using liquid nitrogen.
Determination of protein concentration
The concentration of TYW1 was determined as described in Chapter 4153.
Preparation of SAM
SAM was prepared as previously described in Chapter 327.
Preparation of tRNA
Soluble RNA was extracted from a YPL207W knockout strain of S. cerevisiae as previously described in Chapter 4153.
Enzyme assays
Activity assays
The assays to determine if the C195S-TYW1 and K41A-TYW1 variants were active were performed in 0.1 M Tris-HCl (pH 8.0) buffer containing 0.1 M potassium chloride, 4 mM DTT, 2 mM SAM, 2 mM pyruvate, 10 mM sodium
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dithionite, 1.5 mM methyl viologen, 200 µg yeast tRNA, and 25µM TYW1 variant.
The reactions were allowed to proceed overnight at 50 °C. The RNA was extracted and digested to nucleosides as described previously27. The resulting nucleotide mixture (60 µL) was injected analyzed via LC-MS as previously described27.
Schiff base trapping
The assays to trap the Schiff base intermediate were performed in 0.1 M
Tris-HCl (pH 8.0) buffer containing 0.1 M KCl, 4 mM DTT, 2 mM SAM, 10 mM
13 13 pyruvate (either 1,2,3- C3-pyruvate or 1- C1-pyruvate), 10 mM sodium dithionite,
1.5 mM methyl viologen, 200 µg yeast tRNA, 10 mM sodium cyanoborohydride or sodium cyanoborodeuteride (freshly prepared), and 100µM C195S TYW1 variant. The reactions were allowed to proceed for 7 hours in the glovebox at room temperature. Following the incubation 20 units of trypsin (bovine pancreas), dissolved in 100 mM Tris-HCl (pH 8.0), was added to each assay and they were incubated at room temperature overnight. The following day DTT was added to a final concentration of 9.5 mM and they were incubated for 45 minutes at 56 °C.
The reactions were allowed to cool to room temperature and 2-iodoacetamide, dissolved in 100 mM ammonium bicarbonate, was added to a final concentration of 23 mM and they were incubated in the dark at room temperature for 30 minutes.
Trypsin and other large molecules were removed by filtration through a PES 10K centrifugal filter and 80 µL was injected onto a Thermo Vanquish UHPLC interfaced with a Thermo LTQ OrbiTrap XL. The analytes were separated on a
Thermo hypersil gold C18 column (150 x 2.1 mm) equilibrated in 0.1% TFA 136
(Fisher Optima LC/MS grade) (solution A). Solution B consisted of acetonitrile:
0.1% TFA (Fisher Optima LC/MS grade). The separation program was as follows with a flow rate of 200 µL/min: 0-1 min, 0% B; 1-6.5 min, 0-30% B; 6.5-6.6 min,
30-100% B; 6.6-9.6 min, 100% B; 9.6-9.7 min, 100-0% B; 9.7-12.7 min, 0% B.
The LTQ OrbiTrap XL was operated in positive ion mode with the FT analyzer set to a resolution of 100,000.
Controls to determine which components were required for the trapping of
13 a Schiff base were carried out as described above in the presence of 1,2,3- C3- pyruvate and either sodium cyanoborohydride or sodium cyanoborodeuteride in the absence of one of the following components SAM, sodium dithionite, sodium cyanoborohydride, pyruvate, TYW1, and tRNA. The resulting protein was analyzed as described above.
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Chapter 6:
Mutagenesis study of the cysteine residues hypothesized to ligate the two iron
sulfur clusters in TYW1
All experiments were designed by A. P. Young and V. Bandarian. All experiments were performed by A. P. Young.
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6.1 Abstract
The radical SAM enzyme, TYW1, catalyzes the formation of imG-14 in tRNAPhe from pyruvate and tRNAPhe containing m1G at position thirty-seven. Two crystal structures of TYW1 identified the presence of two cysteine motifs that were close together in the secondary structure of the enzyme. One of the cysteine motifs had the sequence CxxxCxxC, which is characteristic of the radical SAM superfamily and binds a 4Fe-4S cluster, and other Cx10xCx10xC which was hypothesized to coordinate a second cluster. A subsequent EPR study confirmed the presence of a second 4Fe-4S cluster. This chapter describes the site directed mutagenesis of the cysteine residues hypothesized to coordinate the two clusters in the M. jannaschii homolog. The mutants were assayed for the ability to produce 4- demethylwyosine containing tRNA and to reductively cleave SAM, forming 5’- deoxyadenosine. The iron and sulfide content of the mutants were also analyzed confirming the presence of two clusters. A possible role for the second 4Fe-4S in
TYW1 cluster is discussed.
6.2 Introduction
Modifications to the standard four RNA bases have been discovered in all types of RNA, in all domains of life1,2. There are currently over one hundred and fifty of them, with the majority found in tRNA1,2. Position thirty-seven of tRNAPhe contains a wyosine derivative, which is a hypermodified base derived from a genetically encoded guanosine residue, in archaea and eukarya17,18,48. The presence of this base has been shown to prevent -1 frameshifting61,65.
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Wyosine bases are derived from a genetically encoded guanosine and are characterized by the presence of a tricyclic base moiety18. The first step in the biosynthesis of all wyosine derivatives is the methylation of guanosine at the N1 position by the SAM dependent methyl transferase TRM5, forming m1G19,20. The second step in the biosynthesis of wyosine derivatives is the condensation of m1G containing tRNAPhe with pyruvate to form imG-14 by the radical SAM enzyme
TYW117,21,27, as shown in Figure 1.4 (page 24). Depending upon the species, a variety of enzymes go on to decorate the imG-14 scaffold to form the final wyosine derivative found in that organism17,45,48.
The radical SAM superfamily is characterized by the presence of a
CxxxCxxC motif22, that coordinates a 4Fe-4S cluster. The 4Fe-4S cluster is coordinated via the three cysteines at three of the irons, with the fourth, uncoordinated iron, termed the unique iron. This 4Fe-4S cluster cycles between the
+1 and +2 oxidation states and is active in the +1 oxidation state23. The unique iron of the cluster ligates a molecule of SAM via the carboxy and amino portions of the methionine portion as shown by X-ray crystallography28,73,75,77,78,82,83,142, EPR30,32,
Mössbauer spectroscopy26,31, and selenium X-ray absorption spectroscopy34,35.
Inner sphere electron transfers from the cluster, when it is in the +1 oxidation state, to SAM causes homolytic cleavage of the 5’ ribose sulfonium bond generating methionine and the highly reactive intermediate dAdo•. The highly reactive dAdo• abstracts a hydrogen atom from the substrate, which could be a small molecule,
RNA molecule, or a protein, to form a substrate radical and dAdoH. The substrate radical can then go on to form new substrate radicals, in its route to form the final
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product. This is shown in Figure 2.2 (page 45)23 The radical SAM superfamily consists of members that do a wide range of chemistry such as DNA repair70, RNA modification27,71, protein modification143, and cofactor biosynthesis67.
A subset of radical SAM enzymes contains more than one Fe-S cluster, with the additional clusters termed auxiliary clusters85,86. The structures of TYW1 from
M. jannaschii and P. horikoshii have been solved via X-ray crystallography and while neither of them contained electron density for Fe-S clusters they both revealed the presence of two cysteine rich motifs24,25. One of the cysteine rich motifs was the standard radical SAM CxxxCxxC motif. The other motif however, which has the sequence Cx10xCx10xC was grouped close enough in the secondary structure to coordinate a second Fe-S cluster as shown in Figure 6.1 with the P. horikoshii structure. One group hypothesized that the cluster was a 2Fe-2S cluster24 and the other that it was a 4Fe-4S cluster25. A sequence alignment revealed that the six cysteines, thought to ligate the two clusters are completely conserved25. The sequence alignment is shown in Figure 1.4 (page 24) and the cysteines that ligate the radical SAM 4Fe-4S cluster are highlighted in blue and the cysteines hypothesized to ligate the second cluster are highlighted in red. A previous study found that when the cysteine residues proposed to make the second Fe-S cluster
(highlighted in red in Figure 1.4, page 24) were individually mutated to alanine
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Figure 6.1 The active site of P. horikoshii TYW1 homolog looking down the channel between the two proposed cluster binding sites. The radical SAM is proposed to be coordinated by the cysteines on the left and the auxiliary cluster by those on the right. The residue numbering used in the figure is that of M. jannaschii for consistency.
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residues, the protein was unable to complement a ∆TYW1 S. cerevisiae in an in vivo complementation assay25.
The auxiliary clusters found in characterized radical SAM enzymes, thus far, have all been 4Fe-4S clusters28,32,88,90-92,116,121, with the exception of BioB which contains a 2Fe-2S cluster69,96. The only enzymes with well characterized roles for their auxiliary clusters are BioB, LipA, RimO, and MiaB32,94,99. These enzymes catalyze sulfur insertions, methylthio transfer in the case of MiaB and
RimO and sulfide insertion in the case of BioB and LipA, with the sulfur atoms derived from their auxiliary clusters. In most cases the enzyme is only capable of a single turnover destroying the cluster in the process32,93,94,99.
In the X-ray crystal structure of the enzyme MoaA, which catalyzes the first step of molybdopterin biosynthesis, the auxiliary 4Fe-4S cluster was interacting with its substrate GTP28. This interaction was subsequently confirmed by an electron nuclear double resonance study in which an interaction between N1 of
GTP and the 4Fe-4S cluster was detected104.
A class of peptide modifying radical SAM enzymes, containing a SPASM domain, have recently been characterized and found to contain at least one auxiliary cluster110. The role of the auxiliary clusters in these enzymes is unknown with a hypothesis proposed in all cases where the cluster accepts an electron from the substrate during catalysis in order quench a potentially harmful radical species. The cluster could then transfer the electron to an external electron acceptor87,92,116,121,122,128. In the case of anaerobic sulfatase maturation enzyme, which performs a two electron oxidation of cysteine to form a formylglycine
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residue within a protein, multiple turnovers have been demonstrated from only one equivalent of reductant. When a flavodoxin/flavodoxin reductase reducing system is used the enzyme is capable of multiple turnovers from the initial reducing equivalent. In this protein there are two auxiliary 4Fe-4S clusters that are proposed to transfer an electron produced during catalysis to the external flavodoxin/flavodoxin reductase system, which can then reduce the radical SAM cluster and allow the catalytic cycle to repeat81,90.
An EPR study of TYW1 revealed that it does have a second 4Fe-4S cluster26. When TYW1 was incubated with pyruvate there was a change in the both the continuous wave EPR and Mössbauer spectrum leading the authors to hypothesize that pyruvate was interacting with the unique iron of the auxiliary 4Fe-
4S cluster26. As described earlier, there is a precedent for substrate interacting with the auxiliary cluster in the case of MoaA28.
In this chapter the cysteine residues that ligate the two 4Fe-4S clusters are identified through site directed mutagenesis. The mutants were assayed for the ability to make both product (imG-14) and cleave SAM to form dAdoH, and their iron and sulfide content were analyzed.
6.3 Results
Six conserved cysteine residues (M. jannaschii numbering 26, 39, 52, 62,
66, and 69) were mutated to alanine residues via site directed mutagenesis. The six purified mutants were all brown in color suggesting the presence of an Fe-S cluster.
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Figure 6.2 The extracted ion chromatogram at m/z 322 when each mutant was incubated in the presence of dithionite, methyl viologen, SAM, pyruvate, and tRNA. The top trace in both columns is that obtained with wild type protein and the bottom trace in each column is in the absence of either pyruvate or TYW1. The left column contains the cysteines hypothesized to ligate the auxiliary cluster while the right column contains those hypothesized to ligate the radical SAM cluster.
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Figure 6.2 shows the extracted ion chromatogram at m/z 322, [M+H]+ imG-14, obtained when tRNA incubated with each mutant in the presence of dithionite, methyl viologen, pyruvate, and SAM is digested to the nucleoside level following the assay. The left column contains the three cysteine mutants that are hypothesized to ligate the auxiliary cluster while the right contains the three hypothesized to ligate the radical SAM cluster, with the wild type sample on the top of both columns. The peak that elutes at 45 minutes corresponds to the imG-14 nucleoside. imG-14 is present in the wild type sample and the reaction containing C39A variant of TYW1. There is no imG-14 peak formed when either pyruvate or TYW1 are omitted. These six cysteine residues were inactive in a previously described in vivo complementation assay25, so it is surprising that the C39A mutant is active. This assay has been repeated with four different protein preparations and the C39A mutant has been active in two of them and inactive in the other two. The other mutants have been inactive in every assay.
The mutants were tested for their ability to produce dAdoH. It has been shown with many radical SAM enzymes that in the presence of reductant the enzyme can typically catalyze the reductive cleavage of SAM to form methionine and dAdo•71,154,168. This is followed by hydrogen atom abstraction from a site other than substrate by dAdo• forming dAdoH. Each mutant was assayed for the production of dAdoH in the presence of SAM, methyl viologen, and dithionite.
Figure 6.3 shows the extracted ion chromatogram at m/z 252, [M+H+] dAdoH, for each of the mutants, wildtype enzyme, and samples containing no SAM, TYW1, or dithionite following a two-hour incubation at 50 °C. The left column contains the
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Figure 6.3 The extracted ion chromatogram at m/z 252 when each mutant was incubated in the presence of dithionite, methyl viologen, and SAM. The top trace in both columns is that obtained with wild type protein and the bottom two traces in the left column are obtained in the absence of protein and SAM. The bottom trace in the right column is that obtained in the absence of dithionite. The left column contains the cysteines hypothesized to ligate the auxiliary cluster while the right column contains the cysteines hypothesized to ligate the radical SAM cluster.
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three cysteine mutants that are hypothesized to ligate the auxiliary cluster while the right contains the three hypothesized to ligate the radical SAM cluster, with the wild type sample on the top of both columns. The left column also contains the minus TYW1 and minus SAM controls, while the right column contains the minus dithionite control. The peak in the wild type at sample 40.5 minutes is dAdoH. The three cysteine mutants hypothesized to ligate the auxiliary cluster; C26A, C39A, and C52A; produce deoxyadenosine, with the C39A mutant actually producing more dAdoH than the wild type enzyme. Both the minus SAM and minus TYW1 samples do not contain any dAdoH. The three cysteine mutants hypothesized to ligate the radical SAM cluster; C62A, C66A, and C69A; all show the presence of a small peak that has a slightly shifted retention time relative to the dAdoH peak in wild type, presumably arising from a contaminant. The minus dithionite sample has a peak that is shifted almost a full minute earlier in retention time indicating that this peak is not dAdoH, but is probably a degradation product of SAM when no dithionite is around to cause cleavage to dAdo• and methionine. Alternatively, this peak could be due to dAdoH produced from protein that purified in the reduced state.
The iron and sulfide content of the mutants and wild type protein were analyzed by ICP-OES and the Beinert method respectively. The results are shown in Table 6.1 and are consistent with the wild type protein and C39A mutant containing two Fe-S clusters while the other mutants contain one Fe-S cluster.
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Mutant Iron (mol per mol of protein) Sulfide (mol per mol of protein) Wild type 7.2 ± 2.1 9.7 ± 1.3 C26A 3.1 ± 1.5 5.9 ± 2.6 C39A 8.1 ± 0.5 8.7 ± 0.7 C52A 2.6 ± 1.1 3.2 ± 1.6 C62A 3.3 ± 2.5 2.1 ± 2.3 C66A 5.2 ± 1.7 4.8 ± 1.2 C69A 4.3 ± 1.3 6.2 ± 2.0
Table 6.1 This table shows the results of the iron and sulfide analysis of the mutants and wild type protein.
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6.4 Discussion
The radical SAM enzyme TYW1 has been shown to catalyze the formation of imG-14 containing tRNA via the condensation of pyruvate and m1G containing tRNA. Utilizing 13C and deuterium labeled pyruvate it was demonstrated that carbons two and three of pyruvate are incorporated into the imG-14 base27, with carbon two becoming the fifth carbon of the imidazoline ring and carbon three forming the methyl group153. It was demonstrated that dAdo• abstracts a hydrogen atom from the methyl group of m1G via the use of a deuterium labeled methyl group153. A mechanism for this transformation is shown in Figure 3.6 on page 76.
Sequence alignments of TYW1, see Figure 1.4 on page 24, identified the presence of two sets of completely conserved cysteines24,25. The two crystal structures of TYW1 revealed that both sets of cysteines were close together in the secondary structure of the protein24,25. An in vivo complementation assay, in a S. cerevisiae ∆TYW1 strain with the M. jannaschii homolog, found the conserved cysteine residues were required for activity25. A subsequent EPR study revealed that there was a second 4Fe-4S cluster, but did not address which cysteines coordinate it26.
In this study six cysteines; C26, C39, C52, C62, C66, and C69; hypothesized to coordinate the two 4Fe-4S clusters were mutated to alanine residues. The results of activity assays performed using these mutants are shown in
Figure 6.2, and only C39A still retained the ability to modify tRNA and make imG-
14. These assays were performed using four different preparations of protein and this was the only mutant that showed activity, and it was active in two of the four
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preparations. One possible explanation for this activity is that another cysteine, or another residue, is substituting for the mutated one and coordinating the cluster.
There is a threonine residue at position forty-nine, see Figure 6.4 for a picture of the auxiliary clusters hypothesized cysteine ligands and threonine 49, that is located close to the hypothesized location of the cluster and the orientation of the residue is such that the hydroxyl is facing towards the cluster near to the position of C39, in the M. jannaschii structure. There is precedent for serine residues substituting for cysteine residues when they have been mutated into cluster binding motifs and the serine mutant was able to coordinate the cluster in such a way that it still had activity169,170, so it could be hypothesized that T49 is substituting for C39 in the structure and coordinating the cluster. The results with the other five mutants were as expected, they were inactive, and confirmed the in vivo complementation results.
The six mutants were tested for their ability to cleave SAM, by looking for dAdoH production. If SAM can bind to the protein, in the presence of dithionite it can be reductively cleaved forming dAdo• which is then quenched by hydrogen atom abstraction, from a source other than substrate, to form dAdoH. Figure 6.3 shows the results of this experiment and mutation of the three residues hypothesized to coordinate the auxiliary cluster did not affect dAdoH production and C39A actually produced more dAdoH then wild type; which is not surprising as that mutant was active. When the three cysteines that coordinated the radical SAM cluster; C62, C66, and C69; were mutated to alanine residues there was no production of dAdoH in any of those mutants. This was expected as the cluster needs to be present in order for SAM to bind to it and subsequently cleave to form
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Figure 6.4 The proposed site of the auxiliary cluster in M. jannaschii TYW1. T39 is located close to C39 and may be substituting for it in the mutant protein.
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dAdo• and methionine. If the cluster has not formed correctly, due to the mutation,
SAM wouldn’t be able to bind and the oxidation properties of the cluster may also be different, causing the cluster to be unable to be reduced or possibly transfer the electron to SAM. There is a small peak present in the extracted ion chromatogram for the C62A, C66A, and C69A mutants, but this peak has a slightly different retention time and is probably not dAdoH for this reason. When dithionite was removed from the reaction there was a large new peak formed. The new peak, retention time forty mins, elutes earlier than the dAdoH indicating that it is likely not dAdoH; however it is possible that some of the protein purifies in a reduced state and this protein is responsible for production of a small amount of dAdoH.
The minus SAM and TYW1 samples both contain no dAdoH, as expected. The dAdoH produced in these assays is a product derived from SAM, so when SAM is omitted from the reaction mixture it cannot be formed. The absence of dAdoH in the no TYW1 sample indicates that it is a protein dependent process.
The iron and sulfide results are shown in Table 6.1. The wild type protein contains 7.2 ± 2.1 mol of iron and 9.7 ± 1.3 mol of sulfide per mol of protein. This is consistent with the presence of two 4Fe-4S clusters. The C39A variant also contained levels of iron and sulfide consistent with the presence of two 4Fe-4S clusters. The other variants all contained levels of iron and sulfide that were about half of that in the wild type and C39A proteins. This is consistent with the other five variants each only containing one 4Fe-4S cluster. The mols of iron and sulfide may not match with the hypothesized numbers for a variety of reasons. For example, there may be clusters other than 4Fe-4S clusters such as 2Fe-2S or 3Fe-
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4S clusters present in the protein, it is common for some of the clusters in radical
SAM enzymes to be incorrect following reconstitution69,91,116. Another factor that could explain the discrepancy could be from iron that is adventitiously bound.
Nevertheless, the iron and sulfide contents are consistent with the hypothesis that
C26, C52, C62, C66, and C69 are coordinating Fe-S clusters. C39 is also most likely involved in coordinating the cluster, due to the absolute conservation of this residue in all homologs of TYW1 and its inactivity in some preps, but due to the inconsistency in the data no definitive conclusion can be reached.
The data presented in this chapter is consistent with the presence of two Fe-
S clusters, as was proposed when the crystal structures were published24,25 and confirmed via EPR26. The data in this chapter also identifies C26, C52, C62, C66, and C69 as residues that are involved in coordination of Fe-S clusters. C39 is also probably coordinating a cluster as was proposed based upon the crystal structure24,25, but due to the inconsistency in the data obtained it is impossible to make that claim based upon the data presented in this chapter.
6.5 Materials and methods
Cloning of TYW1
The cloning of M. jannaschii TYW1 was previously described in Chapter
327.
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Construction of TYW1 cysteine variants
The M. jannaschii TYW1 cysteine variants were constructed using the
Stratagene QuickChange site-directed mutagenesis protocol, with the primers shown in Table 6.2, and TYW1 pET28a construct as a template.
Expression of TYW1 variants
TYW1, both wild type and variants, were grown and expressed as previously described in Chapter 327.
Purification of TYW1 variants
TYW1, both wild type and variants, were purified as described in Chapter
4153.
Determination of protein concentration
TYW1 concentration was determined as previously described in Chapter
4153.
Preparation of SAM
SAM was prepared as described in Chapter 327.
Preparation of tRNA
Soluble RNA was extracted from a YPL207w deletion strain of S. cerevisiae as previously described in Chapter 4153.
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TYW1 Primer Sequence variant C26A 5’-CCGCAGTGAAACTGGCCGGTTGGGTTCG 5’-CGAACCCAACCGGCCAGTTTCACTGCGG C39A 5’-GGAAGACAAAAACGCTTACAAATCAAAATTC 5’-GAATTTTGATTTGTAAGCGTTTTTGTCTTCC C52A 5’-CGAAACCCACCGCGCCATTCAGTGTACG 5’-CGTACACTGAATGGCGCGGTGGGTTTCG C62A 5’-CGTCGGTTATCTGGGCCCAGCAAAATTGC 5’-GCAATTTTGCTGGGCCCAGATAACCGACG C66A 5’-GGTGCCAGCAAAATGCCATTTTCTGTTGG 5’-CCAACAGAAAATGGCATTTTGCTGGCACC C69A 5’-AAAATTGCATTTTCGCTTGGCGTGTCCTGCC 5’-GGCAGGACACGCCAAGCGAAAATGCAATTTT
Table 6.2 This table shows the primers used to make the alanine variants if the cluster binding cysteines. The residues that were mutated are shown in bold.
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Determination of iron and sulfide stoichiometry
Iron content of TYW1 and the variants was determined by ICP-OES.
Protein was diluted to 1 µM in 1% (v/v) trace metals grade nitric acid. Analysis was conducted by the Analytical Facilities of the Department of Hydrology and
Atmospheric Sciences at the University of Arizona. Acid labile sulfide content of
TYW1 and the variants was determined using the Beinert method171.
Activity assays
The assays to determine if the cysteine variants were active were performed in 0.1 M Tris-HCl (pH 8) buffer containing 0.1 M potassium chloride, 2 mM SAM,
2 mM pyruvate, 1.5 mM methyl viologen, 10 mM sodium dithionite, 4 mM DTT,
100 µg yeast tRNA, and 25 µM TYW1 variant. The assays were incubated overnight at 50°C. The RNA was extracted and digested to nucleosides as described previously27. The resulting nucleotide mixture (60 µL) was analyzed via LC-MS as previously described in Chapter 4153.
The assays to determine if the cysteine variants could produce dAdoH were performed in 0.1 M Tris-HCl (pH 8) buffer containing 0.1 M potassium chloride, 2 mM SAM, 1.5 mM methyl viologen, 10 mM sodium dithionite, 4 mM DTT, and
25 µM TYW1 variant (100 µL total volume). The reactions were quenched by addition of 10 µL of 30% TCA (w/v). Protein was removed by centrifugation and
50 µL was analyzed via LC-MS using the separation program previously described in Chapter 4153.
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Chapter 7:
Conclusions and future work
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TYW1 was initially identified as an enzyme involved in the biosynthesis of yW in S. cerevisiae via reverse genetic approaches. Two separate groups identified that S. cerevisiae strains containing deletions of the gene encoding TYW1
(YPL207w) were unable to produce yW17,21. The role of TYW1 was confirmed via in vivo complementation assays in S. cerevisiae17,21,25. Further studies showed that
TYW1 catalyzed the conversion of m1G to imG-14 in phenylalanine containing tRNA17. Further study of TYW1 was hampered due to the unknown identity of its substrate.
7.1 Conclusions
This dissertation has focused upon the M. jannaschii homolog of the radical
SAM enzyme TYW1. Chapter 3 details the discovery of TYW1s third substrate, the other two being SAM and tRNAPhe containing a m1G modification. Of four substrates tested; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; only the presence of pyruvate led to the conversion of m1G to imG-14.
To confirm the identity of the substrate as pyruvate and to also identify which two carbons of the three carbon pyruvate were incorporated into imG-14 the experiment was performed with 13C isotopologues of pyruvate. The experiments conducted with 13C labeled pyruvate revealed that carbons two and three are incorporated into imG-14, with carbon one lost as a byproduct. A mechanism for this transformation was proposed, Figure 3.6 on page 76, and subsequently tested27.
In Chapter 4 the site of hydrogen atom abstraction by TYW1 was identified.
The hypothesized mechanism, Figure 3.6 on page 76, proposed that hydrogen atom
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abstraction by dAdo• occurs at the m1G methyl group. tRNA containing a deuterated methyl group was used to confirm the methyl group as the site of hydrogen atom abstraction by dAdo• by analyzing the dAdoH produced when both protiated and deuterated tRNA was used. In the presence of deuterated tRNA there was an 200 fold increase in the dAdoH 2H isotope peak when the dAdoH produced was analyzed by high resolution mass spectrometry. The regiochemistry of the addition of carbons two and three of pyruvate to m1G was also identified via the use of deuterium labeled pyruvate. Pyruvate labeled with deuterium at carbon three was used as substrate and showed full retention of all three deuterium atoms in the imG-14 produced, showing that carbon three of pyruvate forms the methyl group of imG-14.
The proposed mechanism, Figure 3.6 on page 76, shows the formation of a
Schiff base between a conserved lysine within the protein and carbon two of pyruvate. Chapter 5 details the trapping of a pyruvate protein adduct that is present on either K41 or K43 (M. jannaschii numbering) and results from reduction of a
Schiff base between pyruvate and the protein. Site directed mutagenesis of K41 produced protein that was inactive, suggesting that this residue may play a role in catalysis. MS/MS fragmentation of the peptide containing the trapped Schiff base is required to identify which residue the pyruvate adduct is part of.
In Chapter 6, six variants of TYW1 were created in which each of the six cysteines that are proposed to coordinate the two Fe-S clusters were mutated to alanine residues. The study found that all of the mutants, with the exception of
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C39A, were inactive and unable to produce imG-14 containing tRNA. A previous in vivo complementation study had found that all six were inactive, and it may be that C39A is active because another residue is substituting for it and coordinating the cluster. It is possible that T49 is fulfilling this role based upon the published crystal structures25,149. The mutants were also assayed for their ability to produce dAdoH. The three mutants that are hypothesized to coordinate the radical SAM
4Fe-4S cluster were unable to produce dAdoH, while the three that are hypothesized to ligate the auxiliary cluster were able to produce dAdoH. Iron and sulfide analysis of these mutants are consistent with both wild type and the C39A mutant containing two 4Fe-4S clusters and the others containing one.
In conclusion, the work described in this dissertation has identified the substrate of TYW1 as pyruvate. Experiments utilizing isotopologues of pyruvate have identified that carbons two and three are incorporated into the RNA base, with carbon one lost as an unidentified byproduct. The regiochemistry of the addition was determined and it was found that carbon three of pyruvate forms the methyl group in imG-14. The site of hydrogen atom abstraction, by dAdo•, was identified as the m1G methyl group using deuterated tRNA substrate. Site directed mutagenesis of K41, the lysine hypothesized to form the Schiff base, to form an alanine variant produced inactive protein. Using NaCNBH3 as a reductant a Schiff base was trapped on a peptide consisting of NCYKSK, residues 38 – 43 of M. jannaschii TYW1. Finally, site directed mutagenesis was used to mutant the hypothesized cluster coordinating cysteines to alanine residues and produced variants that were inactive, with the exception of C39A where another residue may
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be compensating for the mutation and coordinating the cluster. The five inactive mutants all contained iron and sulfide levels consistent with the presence of one cluster, while wild type and the active mutant were consistent with two clusters.
7.2 Future directions
7.2.a Archaeal homologs of TYW1
The next logical step in the study of archaeal homologs of TYW1 would be to identify the role of the auxiliary cluster. This cluster has been hypothesized to coordinate pyruvate26. If this were the case, it could be investigated using the same approach as that in Chapter 5. In Chapter 5 it was demonstrated that it is possible to trap a covalent TYW1 pyruvate complex thorough the use of NaCNBH3. If the cluster plays a role in coordinating pyruvate, carrying out that experiment using the
C26A, C39A, and C52A variants should produce a peptide fragment without the pyruvate adduct. If the TYW1 pyruvate complex were still present in the mutants it would suggest that there is no interaction between the cluster and that some other factor causes pyruvate to bind to TYW1.
Another role for the cluster may be in electron transfer. The mechanism in
Figure 3.6 (page 76) proposes that a carbon dioxide anion radical species is formed during the reaction. If pyruvate were coordinated or interacting with the auxiliary cluster, the cluster may be able to resolve this radical species. Addition of an electron from the cluster to the carbon dioxide anion radical species, along with a proton would produce formate as a final product and the cluster would have been oxidized during the course of the reaction. Alternatively, the cluster could accept
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an electron during the reaction causing carbon dioxide to be produced as the final product and the cluster would be reduced during the course of the reaction. The
EPR study proposed the auxiliary cluster cycled between the +1 and +2 oxidation states in the presence of dithionite26. This would be consistent with the production of formate as the final product. In order to definitively identify the role of the cluster, the identity of the product formed by C1 of pyruvate needs to be established.
7.2.b Eukaryal homologs of TYW1
All radical SAM enzymes require the presence of a reductant, in order for the Fe-S cluster to be in the correct oxidation state for catalysis23,154,168. In the studies detailed above dithionite was used as a reductant in all the experiments, but this is not a biologically relevant reductant. In bacterial homologs of radical SAM enzymes it has been proposed that flavodoxin / flavodoxin reductase is the relevant biological reducing system71,128,168, and has been shown to greatly increase enzyme activity when used in place of dithionite168.
Eukaroytic homologs of TYW1 contain two distinct domains, while archaeal homologs only contain the single radical SAM domain. Eukaroytic homologs of TYW1 contain an N-terminal flavodoxin like domain in addition to the radical SAM domain17. It is proposed that this domain acts as the enzymes source of reductant in order to reduce the Fe-S clusters to the correct oxidation state for catalysis17. Characterization of TYW1 homologs containing this domain would be important to the radical SAM field as this domain may provide evidence of how
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the reducing proteins bind to the radical SAM protein and subsequently reduce the clusters. It may also provide insight into the role of the auxiliary cluster within
TYW1. It is possible that the auxiliary cluster accepts an electron from the radical species proposed to form during the reaction26,27, which is then transferred to the radical SAM cluster allowing catalysis to continue without the input of more electrons from reductant. The use of chemical reductants may prevent this transfer as they could cause the auxiliary cluster to be in an incorrect oxidation state. It would be important to characterize the flow of electrons through the reaction catalyzed by TYW1 with a biologically relevant reducing system and the eukaryotic homologs may provide the opportunity for this.
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Appendix A:
Radical mediated ring formation in the biosynthesis of the hypermodified
tRNA base wybutosine
Reproduced with permission from: Young A. P. and Bandarian V. (2013) Radical mediated ring formation in the biosynthesis of the hypermodified tRNA base wybutosine, Current Opinions in Chemical Biology 17, 613-618.
The manuscript was written by A. P. Young and V. Bandarian.
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166
167
168
169
170
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