J A Krzycki

The Ohio State University, Columbus, Ohio, United States

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Publications (60)391.67 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.
    Proceedings of the National Academy of Sciences of the United States of America. 10/2014;
  • Joseph A Krzycki
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    ABSTRACT: Pyrrolysine is the 22nd genetically encoded amino acid. For many years, its biosynthesis has been primarily a matter for conjecture. Recently, a pathway for the synthesis of pyrrolysine from two molecules of lysine was outlined in which a radical SAM enzyme acts as a lysine mutase to generate a methylated ornithine from lysine, which is then ligated to form an amide with the ɛ-amine of a second lysine. Oxidation of the isopeptide gives rise to pyrrolysine. Mechanisms have been proposed for both the mutase and the ligase, and structures now exist for each, setting the stage for a more detailed understanding of how pyrrolysine is synthesized and functions in bacteria and archaea.
    Current opinion in chemical biology 07/2013; · 8.30 Impact Factor
  • Ruisheng Jiang, Joseph A Krzycki
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    ABSTRACT: Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA(Pyl). Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA(Pyl) in EMSA with apparent K(d) values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA(Pyl) binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA(Pyl) binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA(Pyl) binding. Further indicating the specificity of PylSn interaction with tRNA(Pyl), substitutions of conserved residues in tRNA(Pyl) in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA(Pyl).
    Journal of Biological Chemistry 07/2012; 287(39):32738-46. · 4.65 Impact Factor
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    Marsha A Gaston, Ruisheng Jiang, Joseph A Krzycki
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    ABSTRACT: In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In Escherichia coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two molecules of lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.
    Current opinion in microbiology 06/2011; 14(3):342-9. · 7.87 Impact Factor
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    ABSTRACT: Pyrrolysine, the twenty-second amino acid found to be encoded in the natural genetic code, is necessary for all of the known pathways by which methane is formed from methylamines. Pyrrolysine comprises a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine. In certain Archaea, three methyltransferases initiate methanogenesis from the various methylamines, and these enzymes are encoded by genes with an in-frame amber codon that is translated as pyrrolysine. Escherichia coli that has been transformed with the pylTSBCD genes from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into proteins. The decoding of UAG as pyrrolysine requires pylT, which produces tRNA(Pyl) (also called tRNA(CUA)), and pylS, which encodes a pyrrolysyl-tRNA synthetase. The pylB, pylC and pylD genes are each required for tRNA-independent pyrrolysine synthesis. Pyrrolysine is the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here we provide genetic and mass spectrometric evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a newly uncovered UAG-encoded amino acid, desmethylpyrrolysine, is made from lysine and exogenous D-ornithine in a pylC-dependent process followed by a pylD-dependent process, but it is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-L-methionine (SAM) protein PylB mediates a lysine mutase reaction that produces 3-methylornithine, which is then ligated to a second molecule of lysine by PylC before oxidation by PylD results in pyrrolysine. The discovery of lysine as the sole precursor to pyrrolysine will further inform discussions of the evolution of the genetic code and amino acid biosynthetic pathways. Furthermore, intermediates of the pathway may provide new avenues by which the pyl system can be exploited to produce recombinant proteins with useful modified residues.
    Nature 03/2011; 471(7340):647-50. · 38.60 Impact Factor
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    ABSTRACT: The family Methanosarcinaceae has an expanded repertoire of growth substrates relative to most other methanogenic archaea. Various methylamines, methylated thiols, and methanol can serve as precursors to both methane and carbon dioxide. These compounds are mobilized into metabolism by methyltransferases that use the growth substrate to methylate a cognate corrinoid protein, which in turn is used as a substrate by a second methyltransferase to methylate Coenzyme M (CoM), forming methyl-SCoM, the precursor to both methane and carbon dioxide. Orthologs of the methyltransferases, as well as the small corrinoid proteins, are found in many archaeal and bacterial genomes. Some of these are homologs of the methylamine methyltransferases predicted to require pyrrolysine, an atypical genetically encoded amino acid, for synthesis. As a resource for the study of these sizable families of proteins, we describe here techniques our laboratories have used for the study of methanogen corrinoid-dependent methyltransferases, focusing especially on isolation and assay techniques useful for various activities of components of the methylamine- and methylthiol-dependent CoM methyltransferase systems.
    Methods in enzymology 01/2011; 494:139-58. · 1.90 Impact Factor
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    Michael Rother, Joseph A Krzycki
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    ABSTRACT: Methanogenic archaea are a group of strictly anaerobic microorganisms characterized by their strict dependence on the process of methanogenesis for energy conservation. Among the archaea, they are also the only known group synthesizing proteins containing selenocysteine or pyrrolysine. All but one of the known archaeal pyrrolysine-containing and all but two of the confirmed archaeal selenocysteine-containing protein are involved in methanogenesis. Synthesis of these proteins proceeds through suppression of translational stop codons but otherwise the two systems are fundamentally different. This paper highlights these differences and summarizes the recent developments in selenocysteine- and pyrrolysine-related research on archaea and aims to put this knowledge into the context of their unique energy metabolism.
    Archaea 01/2010; 2010. · 2.55 Impact Factor
  • Joseph A. Krzycki
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    ABSTRACT: Pyrrolysine followed selenocysteine in order of discovery. While both atypical amino acids are encoded by canonical stop codons, the mechanisms by which they are inserted into protein are very different. Pyrrolysine is carried to the ribosome by tRNAPyl (encoded by pylT) whose unusual structure possesses the CUA anticodon needed to decode UAG. A pyrrolysyl-tRNA synthetase (product of pylS) ligates pyrrolysine to tRNAPyl. Pyrrolysine is made by the products of the pylBCD genes without the need for tRNAPyl, contrasting with selenocysteine synthesis on tRNASec. Isolated examples of the pylTSBCD genes, often in a single cluster, have been found in genomes of methanogenic Archaea, G+ Bacteria, and δ-proteobacteria. Escherichia coli transformed with pyl genes translates UAG as endogenously synthesized pyrrolysine. The ease of the lateral transfer of the genetic encoding of pyrrolysine is now being exploited for tailoring recombinant proteins. Pyrrolysine incorporation appears to occur to some extent by amber suppression on a genome-wide basis in methanogenic Archaea. With some methylamine methyltransferase transcripts, a putative pyrrolysine insertion sequence (PYLIS) forms an in-frame stem-loop 3′ to the translated UAG, analogous to such loops required in Bacteria for translation of UGA as selenocysteine. PYLIS sequences are not found in all types of methylamine methyltransferases. Unlike the precedent of selenocysteine, after deletion of PYLIS, significant UAG translation remains with a marked increase in UAG-directed termination, suggesting some part of the PYLIS sequence functions in enhancing amber suppression. Some methanogen genomes encode additional homologs of elongation and release factors, however, their limited distribution suggests at best a nonessential role in enhancing UAG translation as pyrrolysine.
    12/2009: pages 53-77;
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    ABSTRACT: The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H2N·CO·NH·CO·NH2) and one mol CO2 per mol cyanuric acid hydrolyzed, as confirmed by 13C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.
    07/2009; 23(6):387-396.
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    ABSTRACT: Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG=UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA(Pyl) for amber decoding as pyrrolysine. PylS and tRNA(Pyl) have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K(m). The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA(Pyl) was similar to in vitro results, with L-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.
    J Mol Biol. 01/2009; 385(4):1156-64.
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    ABSTRACT: Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the component from the CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and HO/OH bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the -subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
    Proceedings of the National Academy of Sciences 01/2009; 105(28). · 9.81 Impact Factor
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    ABSTRACT: Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG=UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA(Pyl) for amber decoding as pyrrolysine. PylS and tRNA(Pyl) have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K(m). The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA(Pyl) was similar to in vitro results, with L-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.
    Journal of Molecular Biology 12/2008; 385(4):1156-64. · 3.91 Impact Factor
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    ABSTRACT: Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.
    Journal of Biological Chemistry 12/2008; 284(4):2285-95. · 4.65 Impact Factor
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    ABSTRACT: Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1A resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.
    Biochemical and Biophysical Research Communications 09/2008; 374(3):470-4. · 2.28 Impact Factor
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    ABSTRACT: Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO(2) oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO(2) interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the alpha(2)epsilon(2) component from the alpha(8)beta(8)gamma(8)delta(8)epsilon(8) CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H(2)O/OH(-) bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the epsilon-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
    Proceedings of the National Academy of Sciences 08/2008; 105(28):9558-63. · 9.81 Impact Factor
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    ABSTRACT: Methanosarcina spp. begin methanogenesis from methylamines with methyltransferases made via the translation of UAG as pyrrolysine. In vitro evidence indicates two possible routes to pyrrolysyl-tRNAPyl. PylS ligates pyrrolysine to tRNAPyl. Alternatively, class I and class II lysyl-tRNA synthetases (LysRS1 and LysRS2) together form lysyl-tRNAPyl, a potential intermediate to pyrrolysyl-tRNAPyl. The unusual possession of both LysRS1 and LysRS2 by Methanosarcina spp. may also reflect differences in catalytic properties. Here we assessed the in vivo relevance of these hypotheses. The lysK and mtmB transcripts, encoding LysRS1 and monomethylamine methyltransferase, were detectable in Methanosarcina barkeri during early log growth on trimethylamine, but not methanol. In contrast, lysS transcript encoding LysRS2 was detectable during log phase with either substrate. Methanosarcina acetivorans strains bearing deletions of lysK or lysS grew normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyl-tRNAPyl. The lysK and lysS genes could not replace pylS in a recombinant system employing tRNAPyl for UAG suppression. The results support an association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine. However, decreased lysyl-tRNALys in the lysS mutant provides a possible rationale for stable transfer of the bacterial lysS gene to methanoarchaea.
    Molecular Microbiology 04/2007; 64(5):1306 - 1318. · 5.03 Impact Factor
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    ABSTRACT: Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.
    Molecular Microbiology 02/2007; 63(1):229-41. · 5.03 Impact Factor
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    ABSTRACT: Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding tRNA(Pyl) that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Archaea but also in a distantly related Gram-positive Bacterium, indicating past horizontal gene transfer of all five genes. Here we show that lateral transfer of pylTSBCD introduces biosynthesis and genetic encoding of pyrrolysine into a naïve organism. PylS-based assays demonstrated that pyrrolysine was biosynthesized in Escherichia coli expressing pylBCD from Methanosarcina acetivorans. Production of pyrrolysine did not require tRNA(Pyl) or PylS. However, when pylTSBCD were coexpressed with mtmB1, encoding the methanogen monomethylamine methyltransferase, UAG was translated as pyrrolysine to produce recombinant monomethylamine methyltransferase. Expression of pylTSBCD also suppressed an amber codon introduced into the E. coli uidA gene. Strains lacking one of the pylBCD genes did not produce pyrrolysine or translate UAG as pyrrolysine. These results indicated that pylBCD gene products biosynthesize pyrrolysine using metabolites common to Bacteria and Archaea and, furthermore, that the pyl gene cluster represents a "genetic code expansion cassette," previously unprecedented in natural organisms, whose transfer allows an existing codon to be translated as a novel endogenously synthesized free amino acid. Analogous cassettes may have served similar functions for other amino acids during the evolutionary expansion of the canonical genetic code.
    Proceedings of the National Academy of Sciences 02/2007; 104(3):1021-6. · 9.81 Impact Factor
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    ABSTRACT: The methyltransferases initiating methanogenesis from trimethylamine, dimethylamine and monomethylamine possess a novel residue, pyrrolysine. Pyrrolysine is the 22nd amino acid, because it is encoded by a single amber (UAG) codon in methylamine methyltransferase transcripts. A dedicated tRNA(CUA) for pyrrolysine, tRNA(Pyl), is charged by a pyrrolysyl-tRNA synthetase with pyrrolysine. As the first step towards the genetic analysis of UAG translation as pyrrolysine, a 761 base-pair genomic segment in Methanosarcina acetivorans containing the pylT gene (encoding tRNA(Pyl)) was deleted and replaced by a puromycin resistance cassette. The DeltappylT mutant lacks detectable tRNA(Pyl), but grows as wild-type on methanol or acetate. Unlike wild-type, the DeltappylT strain cannot grow on any methylamine, nor use monomethylamine as sole nitrogen source. Wild-type cells, but not DeltappylT, have monomethylamine methyltransferase activity during growth on methanol. Immunoblot analysis indicated monomethylamine methyltransferase was absent in DeltappylT. The phenotype of DeltappylT reveals the deficiency in methylamine metabolism expected of a Methanosarcina species unable to decode UAG codons as pyrrolysine, but also that loss of pylT does not compromise growth on other substrates. These results indicate that in-depth genetic analysis of UAG translation as pyrrolysine is feasible, as deletion of pylT is conditionally lethal depending on growth substrate.
    Molecular Microbiology 02/2006; 59(1):56-66. · 5.03 Impact Factor
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    Joseph A Krzycki
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    ABSTRACT: Pyrrolysine is an amino acid encoded by the amber codon in genes required for methylamine utilization by members of the Methanosarcinaceae. Pyrrolysine and selenocysteine share the distinction of being the only two non-canonical amino acids that have entered natural genetic codes. Recent experiments have shown that encoding of pyrrolysine, unlike that of selenocysteine, also shares an important trait of the original set of twenty amino acids. UAG is translated as pyrrolysine with the participation of a dedicated aminoacyl-tRNA synthetase. Expression of the genes encoding the pyrrolysyl-tRNA synthetase and its cognate tRNA is sufficient to add pyrrolysine to the genetic code of a recombinant organism. Thus, the recruitment of pyrrolysine into the genetic code involved evolution of the first non-canonical aminoacyl-tRNA synthetase and cognate tRNA to be described from nature.
    Current Opinion in Microbiology 01/2006; 8(6):706-12. · 8.23 Impact Factor

Publication Stats

2k Citations
391.67 Total Impact Points

Institutions

  • 1991–2013
    • The Ohio State University
      • • Department of Microbiology
      • • Departments of Chemistry and Biochemistry
      Columbus, Ohio, United States
  • 2011
    • University of Miami
      • Department of Microbiology & Immunology
      Coral Gables, FL, United States
  • 2010
    • Goethe-Universität Frankfurt am Main
      • Institut für Molekulare Biowissenschaften
      Frankfurt am Main, Hesse, Germany
  • 1987
    • Michigan State University
      East Lansing, Michigan, United States
  • 1980–1987
    • University of Wisconsin, Madison
      • Department of Bacteriology
      Mississippi, United States