The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases

Soares, Jitesh A.; Zhang, Liwen; Pitsch, Rhonda L.; Kleinholz, Nanette M.; Jones, R. Benjamin; Wolff, Jeremy J.; Amster, I. Jonathan; Green‐Church, Kari B.; Krzycki, Joseph A.

doi:10.1074/jbc.m506402200

Cited by 61 publications

(62 citation statements)

References 31 publications

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“…Crystallography of MtmB1, the monomethylamine methyltransferase, demonstrated that the UAG codon of the encoding mtmB1 gene corresponds to pyrrolysine, whose structure was proposed as lysine with N in amide linkage with the D-isomer of 4-methyl-pyrroline-5-carboxylate (4,5). This structure is consistent with the UAG-encoded residue's mass in three distinct methylamine methyltransferases (6).…”

supporting

confidence: 57%

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A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Longstaff

Larue

Faust

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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.T ranslated in-frame amber (TAG/UAG) codons in the genes encoding methylamine methyltransferases from Methanosarcina barkeri (1-3) were early clues to the existence of pyrrolysine as the 22nd genetically encoded amino acid from nature. Crystallography of MtmB1, the monomethylamine methyltransferase, demonstrated that the UAG codon of the encoding mtmB1 gene corresponds to pyrrolysine, whose structure was proposed as lysine with N in amide linkage with the D-isomer of 4-methyl-pyrroline-5-carboxylate (4, 5). This structure is consistent with the UAG-encoded residue's mass in three distinct methylamine methyltransferases (6).Near the mtmB1 gene in Methanosarcina spp. are the pyl genes (ref. 7; Fig. 1). Two of these genes are known to underlie the genetic encoding of pyrrolysine. The pylT encodes tRNA Pyl , whose CUA anticodon complements the UAG codon corresponding to pyrrolysine. Deletion of the pyl gene promoter and pylT leads to loss of pyrrolysine-dependent monomethylamine methyltransferase and methylamine metabolism (8). The pylS gene encodes the pyrrolysyl-tRNA synthetase, PylS, which charges tRNA Pyl with chemically synthesized pyrrolysine and further carries out a pyrrolysine-dependent ATP:pyrophosphate exchange reaction (5, 9-11)...

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confidence: 57%

“…The mtmB1 UAG translation product was solubilized in 7 M urea as detected by immunoblotting, and this fraction was further separated by SDS gel electrophoresis. The 50-kDa MtmB1 was subjected to in-gel chymotrypsin digestion and peptide sequencing by tandem mass spectrometry by using previously described methods (6,9).…”

Section: Methodsmentioning

confidence: 99%

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Longstaff

Larue

Faust

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…However, only genes encoding one small clade within this large family of proteins (Fig. 5) have the pyrrolysine codon that is a hallmark of bona fide TMA methyltransferases (30,39). This ambiguity has left the function of the non-Pyl MttB proteins an open question.…”

Section: Discussionmentioning

confidence: 99%

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

Ticak

Kountz²,

Girosky³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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157

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Significance Pyrrolysine, the 22nd amino acid, is found in few proteins. One, the trimethylamine methyltransferase MttB, forms a small portion of a large family of proteins. Most in this family lack pyrrolysine and have no known activity. We show that one such protein, MtgB, is a glycine betaine methyltransferase, providing functional context that may explain the relationship between family members with and without pyrrolysine. Close relatives of MtgB are encoded in many of the abundant bacteria in the oceans, as well as different microbes undertaking symbioses ranging from plants to humans. This finding implies that MtgB might partake in a widespread and underappreciated pathway of GB metabolism contributing significantly to global carbon and nitrogen cycling as well as human health.

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“…In the Methanosarcinaceae, a family of methanogenic archaea, the new amino acid pyrrolysine (Pyl) was shown to be present in the mono-, di-and tri-methylamine methyltransferases (MtmB, MtbB and MttB) [1,2]. This new amino acid is co-translationally inserted in response to an in-frame UAG codon located in the corresponding mRNAs [3].…”

Section: Introductionmentioning

confidence: 99%

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

et al. 2006

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In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA Pyl by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA Pyl . We report here the in vitro aminoacylation of tRNA Pyl by PylRS with two Pyl analogues: -prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L L-lysine (Cyc). Escherichia coli, transformed with the tRNA Pyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of D D-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA Pyl and D D-prolyl-lysine-tRNA Pyl as substrates during protein synthesis. Furthermore, the formation of active b-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.

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The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases

Cited by 61 publications

References 31 publications

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

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