In Vitro Phosphinate Methylation by PhpK from <i>Kitasatospora phosalacinea</i>

Werner, Williard J.; Allen, Kylie; Hu, Kaifeng; Helms, Gregory L.; Chen, Brian S.; Wang, Susan C.

doi:10.1021/bi201220r

Cited by 64 publications

(96 citation statements)

References 30 publications

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“…A subgroup of recently characterized radical SAM methyltransferases involved in secondary metabolite biosynthesis in bacteria contain a vitamin B 12 (cobalamin)-binding motif and require cobalamin for activity (13,(31)(32)(33)(34). These enzymes are thought to use one molecule of SAM in typical radical SAM fashion to produce Ado-CH 2 · for substrate radical formation (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Allen

White

2014

J Bacteriol

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Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX 3 CX 2 C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.

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Section: Resultsmentioning

confidence: 99%

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Allen

White

2014

J Bacteriol

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“…They are distinguished in architecture from other classes of RS methylases by an N-terminal cobalamin-binding domain in addition to the core RS domain. Although predicted cobalamin-containing methylases were the first RS methylases to be recognized (14), their characterization has lagged considerably behind Class A enzymes due to the difficulty associated with their heterologous overproduction in a soluble form (42)(43)(44). Of the four Class B enzymes that have been studied in vitro (TsrM, GenK, Fom3, and PhpK), only TsrM was not purified from insoluble inclusion bodies and refolded before reconstitution of its Fe/S cluster (45).…”

Section: Class B Rs Methylasesmentioning

confidence: 99%

“…PhpK was the first Class B RS methylase to be purified and characterized in vitro (42). It catalyzes the methylation of the phosphinate group of 2-acetylamino-4-hydroxyphosphinylbutanoate (NAcDMPT) to produce 2-acetylamino-4-hydroxymethylphosphinylbutanoate (NAcPT), representing the so-called P-methylase family of RS enzymes (11,46,47).…”

Section: Class B Rs Methylasesmentioning

confidence: 99%

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Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

Bauerle

Schwalm

Booker

2015

Journal of Biological Chemistry

205

236

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Radical S-adenosylmethionine (SAM) enzymes use the oxidizing power of a 5-deoxyadenosyl 5-radical to initiate an amazing array of transformations, usually through the abstraction of a target substrate hydrogen atom. A common reaction of radical SAM (RS) enzymes is the methylation of unactivated carbon or phosphorous atoms found in numerous primary and secondary metabolites, as well as in proteins, sugars, lipids, and RNA. However, neither the chemical mechanisms by which these unactivated atoms obtain methyl groups nor the actual methyl donors are conserved. In fact, RS methylases have been grouped into three classes based on protein architecture, cofactor requirement, and predicted mechanism of catalysis. Class A methylases use two cysteine residues to methylate sp 2 -hybridized carbon centers. Class B methylases require a cobalamin cofactor to methylate both sp 2 -hybridized and sp 3 -hybridized carbon centers as well as phosphinate phosphorous atoms. Class C methylases share significant sequence homology with the RS enzyme, HemN, and may bind two SAM molecules simultaneously to methylate sp 2 -hybridized carbon centers. Lastly, we describe a new class of recently discovered RS methylases. These Class D methylases, unlike Class A, B, and C enzymes, which use SAM as the source of the donated methyl carbon, are proposed to methylate sp 2 -hybridized carbon centers using methylenetetrahydrofolate as the source of the appended methyl carbon.

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“…2). Two genes encoding a B12-dependent radical S-adenosyl methionine (SAM) 10,11) enzyme (res4) and a methyl transferase (res3), both of which would participate in biosynthesis of the isopropyl moiety of 2, were located next to res5. Two ABC transporter 12) genes (res1 and 2) that may participate in efflux of 1 also existed.…”

Section: Resultsmentioning

confidence: 99%

Identification and analysis of the resorcinomycin biosynthetic gene cluster

Ooya

Ogasawara

Noike

2015

Bioscience, Biotechnology, and Biochemistry

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Resorcinomycin (1) is composed of a nonproteinogenic amino acid, (S)-2-(3,5-dihydroxy-4-isopropylphenyl)-2-guanidinoacetic acid (2), and glycine. A biosynthetic gene cluster was identified in a genome database of Streptoverticillium roseoverticillatum by searching for orthologs of the genes responsible for biosynthesis of pheganomycin (3), which possesses a (2)-derivative at its N-terminus. The cluster contained a gene encoding an ATP-grasp-ligase (res5), which was suggested to catalyze the peptide bond formation between 2 and glycine. A res5-deletion mutant lost 1 productivity but accumulated 2 in the culture broth. However, recombinant RES5 did not show catalytic activity to form 1 with 2 and glycine as substrates. Moreover, heterologous expression of the cluster resulted in accumulation of only 2 and no production of 1 was observed. These results suggested that a peptide with glycine at its N-terminus may be used as a nucleophile and then maturated by a peptidase encoded by a gene outside of the cluster.

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In Vitro Phosphinate Methylation by PhpK from Kitasatospora phosalacinea

Cited by 64 publications

References 30 publications

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

Identification and analysis of the resorcinomycin biosynthetic gene cluster

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