COMICR-868; NO. OF PAGES 8 Available online at www.sciencedirect.com Functional context, biosynthesis, and genetic encoding of pyrrolysine Marsha A Gaston1, Ruisheng Jiang2 and Joseph A Krzycki1,2 In Methanosarcina spp., amber codons in methylamine methylate a cognate corrinoid protein (MttC, MtbC, or methyltransferase genes are translated as the 22nd amino acid, MtmC) in the Co(I) state. Adventitious oxidation of the pyrrolysine. The responsible pyl genes plus amber-codon corrinoid proteins to Co(II) can inactivate the methyltrans- containing methyltransferase genes have been identified in four ferase reactions, but the iron-sulfur protein RamA can archaeal and five bacterial genera, including one human activate the corrinoid proteins via ATP dependent pathogen. In Escherichia coli, the recombinant pylBCD gene reduction [7]. The methyl-Co(III) corrinoid proteins products biosynthesize pyrrolysine from two molecules of are substrates of MtbA which methylates coenzyme M lysine and the pylTS gene products direct pyrrolysine (CoM) [8,9]. From here, methyl groups can be converted to incorporation into protein. In the proposed biosynthetic carbon dioxide, methane, and cell carbon. pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by MttB, MtbB, or MtmB have no significant sequence PylD. Structures of the catalytic domain of pyrrolysyl-tRNA similarity, but each of their genes contains a single in- synthetase (archaeal PylS or bacterial PylSc) revealed binding frame amber codon [10,11] that is translated [12,13]. The sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now crystal structure of MtmB revealed pyrrolysine as the being exploited as an orthogonal pair in recombinant systems UAG-encoded residue [3,14]. Mass spectral studies for introduction of useful modified amino acids into proteins. demonstrated that the UAG-encoded residues of MttB Addresses and MtbB are also pyrrolysine [13]. Pyrrolysine was 1 Department of Microbiology, 484 West 12th Avenue, The Ohio State observed in the crystal structure to bind ammonia at University, Columbus, OH 43210, United States the carbon of the imine bond [3,14], and it is hypothesized 2 The Ohio State University Biochemistry Program, 484 West 12th that a pyrrolysine–methylammonium adduct serves to Avenue, The Ohio State University, Columbus, OH 43210, United States activate and orient methylamines as substrates for nucleo- Corresponding author: Krzycki, Joseph A ([email protected]) philic attack by the Co(I) corrinoid protein (Figure 1) [3,15]. Current Opinion in Microbiology 2011, 14:1–8 Pyrrolysine is synthesized and incorporated into the meth- This review comes from a themed issue on ylamine methyltransferases through the combined actions Archaea of the products of the pyl genes (Figure 1). The pylT gene Edited by John Reeve and Christa Schleper encodes tRNAPyl whose CUA anticodon allows for amber codon translation [2,16]. The pylS gene produces the pyrrolysyl-tRNA synthetase that charges tRNAPyl directly 1369-5274/$ – see front matter with pyrrolysine [17,18]. The synthesis of pyrrolysine is # 2011 Elsevier Ltd. All rights reserved. carried out by the pylBCD gene products [19]. DOI 10.1016/j.mib.2011.04.001 Incorporation of pyrrolysine into protein under the direction of amber codons does not require specific signals in the gene, as in-frame amber codons inserted Introduction into the Escherichia coli uidA gene for b-glucuronidase are Methanogenesis is a process unique to the Archaea. Befit- translated as pyrrolysine at 20–30% efficiency in Metha- tingly, many unusual enzymes, cofactors, and metabolites nosarcina acetivorans [20,21 ]. Translation of UAG as were first encountered in methanogens and only later in pyrrolysine even in foreign genes with an introduced bacteria [1]. One such find is pyrrolysine, the 22nd amino amber codon suggests this level of translation occurs by a acid to be encountered in the natural genetic code [2,3]. mechanism analogous to that underlying amber suppres- sion, with tRNAPyl acting as a suppressor tRNA. On the In Archaea, the pyrrolysyl residue is known to occur only in other hand, UAG translation in the methanogen with the family Methanosarcinaceae. Unlike most other methano- mtmB1 transcripts appears much more efficient, with gens, members of this group can use methylamines as little UAG-termination product detectable. Substitution precursors to methane. Metabolism of trimethylamine, of sequence immediately downstream of the UAG codon dimethylamine, or monomethylamine is respectively dramatically increases the UAG-termination product, initiated by the pyrrolysine-containing proteins MttB, but with still a relatively high level of UAG translation MtbB, or MtmB (Figure 1)[4–6]. These proteins each as pyrrolysine. While the deleted region may act as a www.sciencedirect.com Current Opinion in Microbiology 2011, 14:1–8 Please cite this article in press as: Gaston MA, et al. Functional context, biosynthesis, and genetic encoding of pyrrolysine, Curr Opin Microbiol (2011), doi:10.1016/j.mib.2011.04.001 COMICR-868; NO. OF PAGES 8 2 Archaea Figure 1 (2R,3R)-3-methylornithinyl- (2R,3R)-3-methylglutamyl- Pyrrolysine (Pyl) (2R,3R)-3-methylornithine N6-lysine 5-semialdehyde-N6-lysine (Z) NH NH NH 2 2 2 (R) N H2N (R) O H2N (R) O O (R) O (R) (R) (R) (R) O PylB OH H2N HN HN HN SAM H O PylD 2 ATP PylC NH H2O 3 (S) NH (S) NH 2 [H] (S) NH (S) 2 2 2 NH2 HO HO HO HO O O O O Lysine (x 2) PylT Pyl mtmB, mtbB, mttB transcripts (tRNAPyl) Translation of UAG as Pyl PylS R + 1 NH CH3 2 CH H N 3 R1 .. R2 N Co (III) HSCoM R2 MtmB MtbB MttB MtmC MtbC MttC R =H R =H R =CH MtbA 1 1 1 3 (MtmB) (MtbB) (MttB) CH4 R2=H R2=CH3 R2=CH3 CH3 R2 R1 N+ CH SCoM CO Co (I) 3 2 H N ATP RamA Cell C - e- e Co (II) Current Opinion in Microbiology Schematic of pyrrolysine and methylamine metabolism in Methanosarcina spp. Pyrrolysine is made from two molecules of lysine. In the proposed pathway, PylB converts one lysine into a methylated D-ornithine derivative, which is then ligated to another lysine by PylC. The resultant dipeptide is oxidized by PylD, which results in spontaneous elimination of water and formation of pyrrolysine. Pyrrolysine is then ligated to tRNAPyl by PylS. The pyrrolysyl-tRNAPyl is carried to the ribosome by the usual elongation factor for cotranslational incorporation into one of the three methylamine methyltransferases, MtmB, MtbB, or MttB. Below each methyltransferase is indicated its particular methylamine substrate. Pyrrolysine in the catalytic site is hypothesized to form an adduct with that methylamine which orients and activates it for methyl group transfer to the Co(I) form of the corrinoid cofactor bound to either MtmC, MtbC, or MttC. Each corrinoid protein interacts preferentially with the methyltransferase indicated below it. Adventitious oxidation of the corrinoid protein can result in inactivation, and a single protein, RamA, can return the corrinoid protein to the Co(I) state. All three methylated corrinoid proteins can serve as substrates for MtbA, which methylates the thiol of CoM (HSCoM). Methyl-CoM can then serve to directly generate methane, or to enter pathways leading to carbon assimilation and carbon dioxide production. Further details are provided in the text. sequence enhancing translation over termination with sequencing has now expanded the count to six bacterial mtmB1 transcripts, previous suggestions from several and six archaeal species belonging to nine genera laboratories of an obligate pyrrolysine insertion element (Figure 2). In the archaea, pyl genes are still limited to or tRNAPyl specific translation factors have not been members of the Methanosarcinaceae, but now also include borne out [20–24]. psychrotrophic methanogen Methanococcoides burtonii, and two halophilic methanogens, Methanohalophilus mahii and Sequenced genomes with pyl genes Methanohalobium evestigatum. The pyl genes are also found At the time of the discovery of pyrrolysine, only Metha- in selected species of the bacterial groups Clostridia or the nosarcina spp. and the bacterium Desulfitobacterium haf- Deltaproteobacteria. The latter group includes symbionts niense were known to possess pyl genes [2]. Recent of multicellular organisms, such as a gut inhabitant of a Current Opinion in Microbiology 2011, 14:1–8 www.sciencedirect.com Please cite this article in press as: Gaston MA, et al. Functional context, biosynthesis, and genetic encoding of pyrrolysine, Curr Opin Microbiol (2011), doi:10.1016/j.mib.2011.04.001 COMICR-868; NO. OF PAGES 8 Functional context, biosynthesis, and genetic encoding of pyrrolysine Gaston, Jiang and Krzycki 3 marine worm identified in a metagenomic study [25,26]. [32]. In bacteria, N-terminal and C-terminal domains of Most recently, pyl genes were annotated in the genome of archaeal PylS are respectively represented by two inde- the human intestinal bacteria, Bilophila wadsworthia pendent proteins, PylSn and PylSc (Figure 2)[2,32]. (Figure 2). The unannotated pylT gene was identified by the authors some distance from the other pyl genes Crystal structures of the C-terminal domain of archaeal (Figure 2, Figure S1). This organism has been a common PylS and bacterial PylSc have revealed the catalytic site isolate in cases of gangrenous appendicitis and abscesses that accommodates pyrrolysine and ATP [30,31,33, in a variety of bodily locations [27], and represents the 34,35]. The pyrrolysine ring is accommodated by a first known human symbiont, and pathogen, to have hydrophobic pocket closed by a mobile loop bearing a pyrrolysine genes. tyrosine which may H-bond the imine nitrogen of pyrro- lysine [30], and/or provide stability to the formed pyrro- While amber codons have been identified in Thg1 and lysyl-adenylate before tRNA binding [33]. While transposase genes of one or two Methanosarcina spp., these pyrrolysine has the most favorable kinetics for amino acid are likely to be mutations stable in the context of tRNAPyl activation, analogs having an oxygen atom replacing the and PylS, as homologs of these genes without amber imine nitrogen are favored over those with carbon, codons exist in other Methanosarcinaceae [21,28,29].