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An enzymatic activation of formaldehyde for nucleotide methylation.

ABSTRACT: Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH₂O-shunt may be relevant in several other important folate-dependent reactions.

SUBMITTER: Bou-Nader C

PROVIDER: S-EPMC8316439 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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An enzymatic activation of formaldehyde for nucleotide methylation.

Bou-Nader Charles C Stull Frederick W FW Pecqueur Ludovic L Simon Philippe P Guérineau Vincent V Royant Antoine A Fontecave Marc M Lombard Murielle M Palfey Bruce A BA Hamdane Djemel D

Nature communications 20210727 1

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for act ...[more]

PMID: 34315871

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Project description:Methylation is an essential and ubiquitous reaction that plays an important role in a wide range of biological processes. Most biological methylations use S-adenosylmethionine (SAM) as the methyl donor and proceed via an S(N)2 displacement mechanism. However, researchers have discovered an increasing number of methylations that involve radical chemistry. The enzymes known to catalyze these reactions all belong to the radical SAM superfamily. This family of enzymes utilizes a specialized [4Fe-4S] cluster for reductive cleavage of SAM to yield a highly reactive 5'-deoxyadenosyl (dAdo) radical. Radical chemistry is then imposed on a variety of organic substrates, leading to a diverse array of transformations. Until recently, researchers had not fully understood how these enzymes employ radical chemistry to mediate a methyl transfer reaction. Sequence analyses reveal that the currently identified radical SAM methyltransferases (RSMTs) can be grouped into three classes, which appear distinct in protein architecture and mechanism. Class A RSMTs mainly include the rRNA methyltransferases RlmN and Cfr from various origins. As exemplified by Escherichia coli RlmN, these proteins have a single canonical radical SAM core domain that includes an (βα)(6) partial barrel most similar to that of pyruvate formate lyase-activase. The exciting recent studies on RlmN and Cfr are beginning to provide insights into the intriguing chemistry of class A RSMTs. These enzymes utilize a methylene radical generated on a unique methylated cysteine residue. However, based on the variety of substrates used by the other classes of RSMTs, alternative mechanisms are likely to be discovered. Class B RSMTs contain a proposed N-terminal cobalamin binding domain in addition to a radical SAM domain at the C-terminus. This class of proteins methylates diverse substrates at inert sp(3) carbons, aromatic heterocycles, and phosphinates, possibly involving a cobalamin-mediated methyl transfer process. Class C RSMTs share significant sequence similarity with coproporphyrinogen III oxidase HemN. Despite methylating similar substrates (aromatic heterocycles), class C RSMTs likely employ a mechanism distinct from that of class A because two conserved cysteines that are required for class A are typically not found in class C RSMTs. Class A and class B enzymes probably share the use of two molecules of SAM: one to generate a dAdo radical and one to provide the methyl group to the substrate. In class A, a cysteine would act as a conduit of the methyl group whereas in class B cobalamin may serve this purpose. Currently no clues are available regarding the mechanism of class C RSMTs, but the sequence similarities between its members and HemN and the observation that HemN binds two SAM molecules suggest that class C enzymes could use two SAM molecules for catalysis. The diverse strategies for using two SAM molecules reflect the rich chemistry of radical-mediated methylation reactions and the remarkable versatility of the radical SAM superfamily.

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An enzymatic activation of formaldehyde for nucleotide methylation.

Publications

An enzymatic activation of formaldehyde for nucleotide methylation.

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