Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

Abstract

TokK and ThnK stereoselectively abstract the pro-S hydrogen, and methylation proceeds with inversion of absolute configuration at C6.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB

“…It was hypothesized that the three observed products (i.e., 18-P , 10-P , and 11-P ) are produced sequentially beginning with C2′-methylation of 2′-dAMP ( 3 → 18-P , Figure A). OxsB then catalyzes dehydrogenation of the C2′-CH 3 methyl to yield the methylidene 10-P rather than a second round of methylation as observed in other radical-mediated methyltransferases. − Although examples of radical SAM catalyzed dehydrogenation of alcohols, thiols, and amines are well-known, − biological C-CH 3 dehydrogenase activity has not been previously reported for a radical SAM enzyme. Nevertheless, NosL from nosiheptide biosynthesis catalyzes C–C desaturation of a substrate structural analogue, and DarE from the darobactin maturation pathway has been identified to catalyze off-pathway dehydrogenation of the C α –C β bond of a tryptophan residue in its peptide substrate. , Once formed, 10-P could then serve as an acceptor for OxsB catalyzed addition of the 5′-deoxyadenosyl radical ( 2 ).…”

“…Moreover, OxsB was also found to be responsible for a series of consecutive reactions beginning with C2′ methylation. In contrast to several other B 12 -dependent radical SAM enzymes, − however, these reactions do not involve repeated methylation at either the same site or the newly introduced methyl substituent resulting in ethyl, iso -propyl, tert -butyl, or sec -butyl groups (Figure S2). Instead, OxsB catalyzes C–C dehydrogenation of the newly installed methyl group followed by radical addition of 5′-deoxyadenosine to the resulting methylidine moiety.…”

Mechanism of S-Adenosyl-l-methionine C-Methylation by Cobalamin-dependent Radical S-Adenosyl-l-methionine Methylase in 1-Amino-2-methylcyclopropanecarboxylic Acid Biosynthesis

“…Crystal structures of Mmp10, which catalyzes C -methylation at the Cδ position of the arginine residue of methyl-coenzyme M reductase, did not elucidate the stereoselectivity of the hydrogen atom abstraction by 5′-dA· . Detailed biochemical analysis of TokK and ThnK with stereoselectively deuterated substrates revealed that inversion of configuration occurs during the TokK and ThnK reactions . In the Orf29 reaction, two molecules of SAM appear to be accommodated precisely in the active site and one molecule of SAM coordinates to the [4Fe-4S] cluster, where it is reductively cleaved to form 5′-dA· and Met ( 6 ).…”

“…26 Detailed biochemical analysis of TokK and ThnK with stereoselectively deuterated substrates revealed that inversion of configuration occurs during the TokK and ThnK reactions. 23 In the Orf29 reaction, two molecules of SAM appear to be accommodated precisely in the active site and one molecule of SAM coordinates to the [4Fe-4S] cluster, where it is reductively cleaved to form 5′-dA• and Met (6). The generated 5′-dA• abstracts the pro-R hydrogen atom of C-4″ of another molecule of SAM to yield the substrate radical and 5′-dA (5).…”

“…In gentamicin biosynthesis, GenK is known to catalyze the C -methylation of the pseudotrisaccharide gentamicin X2 at the C-6′ position to yield Geneticin (G418); this reaction also proceeds with retention of stereochemistry . However, most MeCbl-dependent radical SAM methylases, including Fom3, − ThnK (and TokK), and GenD1, catalyze C -methylation with the inversion of configuration at the methylation positions. The crystal structure of TokK complexed with substrate, 5′-dA, and hydroxocobalamin (OHCbl) revealed that C-5′ of 5′-dA is located at a similar distance from both geminal hydrogen atoms of the substrate at the reactive site .…”