Mechanistic Studies on CysS – A Vitamin B₁₂-Dependent Radical SAM Methyltransferase Involved in the Biosynthesis of the tert-Butyl Group of Cystobactamid

Abstract

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) methyltransferases catalyze methylation reactions at non-nucleophilic centers in a wide range of substrates. CysS is a Cbldependent radical SAM methyltransferase involved in cystobactamid biosynthesis. This enzyme catalyzes the sequential methylation of a methoxy group to form ethoxy, i-propoxy, s-butoxy, and t-butoxy groups on a p-aminobenzoate peptidyl carrier protein thioester intermediate. This biosynthetic strategy enables the host myxobacterium to biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogues. In this Article, we describe three experiments to elucidate how CysS uses Cbl, SAM, and a [4Fe−4S] cluster to catalyze iterative methylation reactions: a cyclopropylcarbinyl rearrangement was used to trap the substrate radical and to estimate the rate of the radical substitution reaction involved in the methyl transfer; a bromoethoxy analogue was used to explore the active site topography; and deuterium isotope effects on the hydrogen atom abstraction by the adenosyl radical were used to investigate the kinetic significance of the hydrogen atom abstraction. On the basis of these experiments, a revised mechanism for CysS is proposed.

Crystallographic snapshots of a B12-dependent radical SAM methyltransferase

“…In addition, a hydrophobic pocket prevents water from converting the pentacoordinated MeCbl into its more stabilized hexacoordinated counterpart, which is a strategy conserved in other B 12 -dependent radical SAM enzymes. These enzymes thus appear to have evolved unique structures and mechanisms to alkylate sp 2 -and sp 3 -hybridized carbon atoms using the twin catalytic power of the cobalamin and SAM cofactors 8,13,14,[16][17][18][19]21,50,51 . In contrast to catalysis by known radical SAM enzymes, catalysis by Mmp10 requires active site reorganization and SAM flexibility within the active site.…”

“…The structure of MCR 3 has revealed several distinct features for this 300-kDa (αβγ) 2 protein complex, such as a unique F 430 cofactor 11 and unusual post-translational modifications 5 , including 5-C-(S)-methylarginine 4,5 , which tunes the reactivity of its active site 6,12 . Mmp10, which has been shown to catalyse this key post-translational modification 7,8 , belongs to an emerging superfamily of B 12 -dependent radical SAM enzymes [13][14][15][16][17][18][19][20][21] that encompasses more than 200,000 proteins (http://radicalsam.org/) 22 . These enzymes are involved in the biosynthesis of myriad natural products including bacteriochlorophyll and antibiotics 9,16,18,23 and catalyse various reactions such as methyl transfer to sp 2 -and sp 3 -hybridized carbon atoms 13,14,18,24 , P-methylation 25 , ring contraction and cyclization reactions 26,27 .…”

“…Mmp10, which has been shown to catalyse this key post-translational modification 7,8 , belongs to an emerging superfamily of B 12 -dependent radical SAM enzymes [13][14][15][16][17][18][19][20][21] that encompasses more than 200,000 proteins (http://radicalsam.org/) 22 . These enzymes are involved in the biosynthesis of myriad natural products including bacteriochlorophyll and antibiotics 9,16,18,23 and catalyse various reactions such as methyl transfer to sp 2 -and sp 3 -hybridized carbon atoms 13,14,18,24 , P-methylation 25 , ring contraction and cyclization reactions 26,27 . However, despite the numerous biochemical and spectroscopic studies available in the literature [13][14][15][16][17][18][19][20][21] , knowledge of these biological catalysts remains limited.…”

“…These enzymes are involved in the biosynthesis of myriad natural products including bacteriochlorophyll and antibiotics 9,16,18,23 and catalyse various reactions such as methyl transfer to sp 2 -and sp 3 -hybridized carbon atoms 13,14,18,24 , P-methylation 25 , ring contraction and cyclization reactions 26,27 . However, despite the numerous biochemical and spectroscopic studies available in the literature [13][14][15][16][17][18][19][20][21] , knowledge of these biological catalysts remains limited. Notably, only two structures of B 12 -dependent radical SAM enzymes have been solved thus far; however, these studies present some limitations precluding a deep understanding of their catalysis 24,27 .…”

A biomimetic S H 2 cross-coupling mechanism for quaternary sp 3 -carbon formation

“…Although cobalamin provides critical stabilization to the reactive methyl radical, the methyl-cobalt bond remains notably weak (bond dissociation energy (BDE) = ~37 kcal/mol), which underpins the kinetic preference for the S H 2 mechanism and heteroselective carbon-carbon bond formation (12). Elegant biosynthetic studies have shown that the rates of such enzymatic S H 2 reactions are extremely fast (~ 10 8 s -1 ) and enable the formation of sterically congested quaternary C(sp 3 ) centers (11). However, despite broad biochemical relevance, S H 2-based cross-coupling paradigms remain effectively unknown within the laboratory setting outside of stoichiometric organonickel methylation or intramolecular S H -cyclizations from seminal contributions of Sanford, Zhang and others (13)(14)(15)(16)(17)(18).…”