Structural Basis of Adomet-Dependent Aminocarboxypropyl Transfer Reaction Catalyzed by Trna-Wybutosine Synthesizing Enzyme, TYW2
Total Page：16
File Type：pdf, Size：1020Kb
Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2 Masataka Umitsua, Hiroshi Nishimasua, Akiko Nomab, Tsutomu Suzukib, Ryuichiro Ishitania,1, and Osamu Nurekia,1 aDepartment of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; and bDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved July 28, 2009 (received for review May 13, 2009) S-adenosylmethionine (AdoMet) is a methyl donor used by a wide ture (4-demethylwyosine, imG-14), using an adenosyl radical variety of methyltransferases, and it is also used as the source of derived from AdoMet (Fig. 1A) (8). Furthermore, three en- an ␣-amino-␣-carboxypropyl (‘‘acp’’) group by several enzymes. zymes, TYW2, 3, and 4, catalyze AdoMet-dependent modifica- tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogen- tions to form the yW nucleoside. In this pathway, TYW2 esis of a hypermodified nucleotide, wybutosine (yW), and it cat- catalyzes the transfer of the ␣-amino-␣-carboxypropyl (acp) alyzes the transfer of the ‘‘acp’’ group from AdoMet to the C7 group, instead of the methyl group, of AdoMet to the C7 position position of the imG-14 base, a yW precursor. This modified nucle- of the tricyclic core structure of imG-14, forming yW-86 (yW oside yW is exclusively located at position 37 of eukaryotic minus 86 Da) (Fig. 1A) (5). A recent study indicated that this acp tRNAPhe, and it ensures the anticodon-codon pairing on the ribo- group of the yW base has a significant negative impact on Ϫ1 somal decoding site. Although this ‘‘acp’’ group has a significant frame shifting at a phenylalanine codon (9), suggesting the role in preventing decoding frame shifts, the mechanism of the biological importance of TYW2 for the maintenance of trans- ‘‘acp’’ group transfer by TYW2 remains unresolved. Here we report lational fidelity. In addition, the enzymes that transfer the acp the crystal structures and functional analyses of two archaeal group of AdoMet to substrates are also present in the biosyn- homologs of TYW2 from Pyrococcus horikoshii and Methanococcus thetic pathways for diphthamide (10), nocardicin (11), and BIOCHEMISTRY jannaschii. The in vitro mass spectrometric and radioisotope-label- diacylglyceryl-O-4Ј-(N,N,N,-trimethyl) homoserine (12). How- ing analyses confirmed that these archaeal TYW2 homologues ever, the detailed mechanism of the acp group transfer by these have the same activity as yeast TYW2. The crystal structures enzymes remains elusive. verified that the archaeal TYW2 contains a canonical class-I meth- Previous studies demonstrated the existence of wyosine (Y) yltransferase (MTase) fold. However, their AdoMet-bound struc- derivatives in several archaea (13–15). Moreover, homologous tures revealed distinctive AdoMet-binding modes, in which the genes to eukaryotic TYW1 and TYW2 exist in archaeal ge- ‘‘acp’’ group, instead of the methyl group, of AdoMet is directed to nomes, suggesting that these Y derivatives are formed by a the substrate binding pocket. Our findings, which were confirmed similar modification pathway to that in eukaryotes. Therefore, by extensive mutagenesis studies, explain why TYW2 transfers the these Y derivatives may play a similar role in maintaining the ‘‘acp’’ group, and not the methyl group, from AdoMet to the translational fidelity of the phenylalanine codon, as in eu- nucleobase. karyotes (15). However, there is no experimental evidence showing that the archaeal Y derivatives are located in the modification ͉ S-adenosylmethionine ͉ X-ray crystallography tRNAPhe anticodon loop. In addition, previous studies reported the archaea-specific Y derivative, 7-methylwyosine (mimG, Fig. ost-transcriptional modifications of RNA molecules have 1A), in which the C7 position is modified by the methyl group, Psignificant biological roles in maintaining life. More than 100 rather than the acp group (13). Thus, the in vivo functions of different modifications exist throughout the three phylogenic these archaeal TYW2 homologues remain elusive. domains (1). Most of these modifications are found in transfer Here, we report the functional characterizations and struc- RNA (tRNA), and they confer various chemical properties to the tural analyses of two archaeal TYW2 enzymes from Pyrococcus RNA residues. Especially, the anticodon loop of tRNA contains horikoshii and Methanococcus jannaschii. An in vitro reconsti- many modified nucleosides, which are considered to facilitate tution assay by mass spectrometry and kinetic analyses with the correct codon-anticodon pairing on the ribosome (2). radioisotope-labeled AdoMet confirmed that the archaeal Wybutosine (yW) is a hypermodified nucleoside located at TYW2 enzymes catalyze the acp group transfer reaction in an position 37, adjacent to the 3Ј position of the anticodon, of AdoMet-dependent manner. The crystal structures of the ar- eukaryotic tRNAPhe. The chemical structure of yW is charac- chaeal TYW2s, in conjunction with the extensive mutational terized by the tricyclic 1H-imidazo[1,2-␣]purine core with a large analyses, provide the structural basis for the transfer of the acp side chain (3) (Fig. 1A). Its bulky, hydrophobic structure is group of AdoMet to the substrate. considered to stabilize not only the conformation of the anti- codon loop itself but also the codon-anticodon pairing on the Ϫ Author contributions: T.S., R.I., and O.N. designed research; M.U., A.N., and R.I. performed ribosome, thereby preventing a potential 1 frame shift at a research; M.U., H.N., A.N., and R.I. analyzed data; and M.U., H.N., A.N., T.S., R.I., and O.N. phenylalanine codon (4). wrote the paper. Recent studies revealed the biosynthetic pathway of yW in The authors declare no conflict of interest. yeast, in which five enzymes catalyze successive S-adenosylme- This article is a PNAS Direct Submission. thionine (AdoMet)-dependent reactions to form yW from the Data deposition: The atomic coordinates and structure factors have been deposited in the G37 residue (5, 6) (Fig. 1A). In the first step, the AdoMet- Protein Data Bank, www.pdb.org (PDB ID codes 3A25, 3A26, and 3A27). 1 dependent tRNA methyltransferase (MTase) Trm5 forms N - 1To whom correspondence may be addressed. E-mail: [email protected] or 1 methylated G37 (m G) (6, 7). In the next step, tRNA-yW [email protected]. synthesizing enzyme-1 (TYW1) catalyzes the radical reaction, This article contains supporting information online at www.pnas.org/cgi/content/full/ involving two [4Fe-4S] clusters, to form the tricyclic ring struc- 0905270106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905270106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Acp-group transfer catalyzed by PhTYW2. (A) Biosynthetic pathway of wybutosine. The chemical structures of the wyosine derivatives are shown. Modification enzymes, Trm5 and TYW1–4, are involved in each modification step. The chemical groups included in each reaction are shadowed in blue. The chemical structure of AdoMet is shown in the box. The methyl and acp groups are colored green and blue, respectively. (B) In vitro reconstitution of yW-86 Phe synthesis using recombinant PhTYW2. RNaseT1-digested yeast tRNA with the imG-14 intermediate (black), obtained from the ⌬TYW2 strain, was reacted with PhTYW2 in the presence (left panel) or absence (right panel) of AdoMet and was analyzed by LC/MS. Mass chromatographs for anticodon-containing fragments including imG-14 (m/z 661, black), yW-86 (m/z 678, green), and yW-173 (m/z 664, red) are overlaid. (C) Kinetic analysis of the acp-group transfer by PhTYW2. The ␣-carboxyl-14C labeled AdoMet and the imG-14 containing tRNAPhe were incubated at 50 °C with recombinant PhTYW2 (closed circles) and without PhTYW2 (open circles), and the incorporation of ␣-carboxyl-14C into the tRNAPhe was quantified by scintillation counting. Data are represented as mean Ϯ SE (n ϭ 3). Results and Discussion spectrometric analyses, a trace amount of yW-173 (MW 335) was Acp-Group Transfer Activity of Archaeal TYW2. The P. horikoshii detected in all products as well as in the substrate, regardless of PH0793 (278 amino acids) and M. jannashii MJ1557 (249 amino the presence of the enzyme or AdoMet (Fig. 1B and Fig. S1). acids) proteins share 22% and 25% sequence identities to yeast This minor peak may stem from a methylated product of imG-14 TYW2, respectively. To determine whether these archaeal at position N4, formed by endogenous TYW3 in yeast cells. TYW2 homologs possess an activity to transfer the acp group to Altogether, our results indicate that the PH0793 and MJ1557 imG-14 at position 37 of tRNAPhe, we performed in vitro proteins have an activity to form yW-86 from imG-14 at position reconstitution assays using these recombinant proteins (Fig. 1B 37 of tRNAPhe. Therefore, we hereafter refer to PH0793 and and Fig. S1). As a substrate, we used tRNAPhe purified from the MJ1557 as PhTYW2 and MjTYW2, respectively. yeast ⌬TYW2 strain, which contains imG-14 at position 37 (Fig. S1A). In the reconstitution assay with the PH0793 and MJ1557 Structure Determination. To understand the mechanism of the proteins, a mass spectrometric analysis detected AdoMet- modification by TYW2, we determined the crystal structures of dependent products corresponding to yW-86 (Fig. 1B and Fig. S1 PhTYW2 in the AdoMet-bound and 5Ј-deoxy-5Ј-methylthioad- B and D). We confirmed our assignment of the peaks by the enosine (MeSAdo)-bound forms, and MjTYW2 in the AdoMet- positive control experiment with purified yeast TYW2 (Fig. bound form (Fig. 2). The initial phases were obtained by the S1C). To further confirm the activity of the archaeal TYW2 molecular replacement method, using the apo-form PhTYW2 homologs, we assayed the acp-group transfer by monitoring the structure as a search model (PDB ID: 2FRN), which was incorporation of the acp group from ␣-carboxyl-14C labeled previously determined by the structural genomics group as a AdoMet into the imG-14 containing tRNAPhe (Fig.