Stereospecific Radical-Mediated B₁₂-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3

Abstract

Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPP-CMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro- R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2 S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.

Genome mining- and synthetic biology-enabled production of hypermodified peptides

“…Cobalamin-dependent rSAM enzymes that methylate unactivated carbon centers are biochemically intriguing, but technically demanding enzymes with barely utilized synthetic potential 48,49 . With more than 7000 members predicted from sequenced genes, they are involved in the biosynthesis of vitamins and many bioactive compounds including carbapenems, gentamicin, fosfomycin, novobiocins, moenomycins, thiostrepton, and bottromycins [50][51][52][53] . Most of the few characterized enzymes catalyze 1-2 C-methylations, and CysS was shown to install up to 3 C-methyl groups into cystobactamid 54 .…”

Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp3 C–H functionalization

“…Whereas many of these approaches rely on the manipulation of functional groups, certain enzymes, including members of the radical S -adenosylmethionine (SAM) family, can perform alkylation of sp 3 C–H bonds. This has been an especially versatile strategy for structural diversification, as seen by its essential role in the biosynthesis of structurally varied natural products and cofactors 4–6 . Known biological machineries for this transformation, however, are limited to enzymes that transfer a methyl group 5,6 or conjugate an activated radical acceptor substrate 4,7 to specific molecules, with methylation as a common mode for sp 3 C–alkyl installation by radical SAM enzymes (Fig.…”

Die Hydrogenobyrsäure‐Struktur enthüllt den Corrin‐Liganden als entatisches Zustandsmodul zur Steigerung der Katalyseaktivität von B12‐Cofaktoren

“…[6,15] In Hby und HCor + brechen die inneren H-Atome jeweils die inhärente C 2 -Symmetrie des Corrin-Kerns,i mG egensatz zur Situation bei den regelmäßiger strukturierten Cobalt-Corrinen und den expandierten und symmetrischen Porphyrinen. [25] Ein ähnlicher Spannungsabbau ist mçglicherweise bei der heterolytischen Abstraktion der cobalt-gebundenen Methylgruppe von MeCbl durch Nukleophile in B 12abhängigen enzymatischen Methyltransfers abzuleiten, die unter Bildung von Co I -Cobalamin ablaufen. Der effektive Koordinationsradius in Hby (1.916 )i st somit nahe an der Länge der äquatorialen Co-N-Bindung in AdoCbl (1.897 ), [17] MeCbl (1.898 ) [18] und Cbl II (1.88 ).…”

“…[4c, 23] Dieselbe Art von Spannung aktiviert die cobalt-gebundene Methylgruppe von MeCbl zur Abstraktion durch Radikale [24] in B 12 -abhängigen Radikal-SAM-Enzymen. [25] Ein ähnlicher Spannungsabbau ist mçglicherweise bei der heterolytischen Abstraktion der cobalt-gebundenen Methylgruppe von MeCbl durch Nukleophile in B 12abhängigen enzymatischen Methyltransfers abzuleiten, die unter Bildung von Co I -Cobalamin ablaufen. [26] In der Adenosyl-Transferase ACAi st eine instabile vierfach koordinierte Form von Cbl II (Cbl II ACA) [20] das kritische Substrat für die Reduktion zur schwer erreichbaren vierfach koordinierten Co I -Spezies.S olchen essentiellen vierfach koordinierten Co II -u nd Co I -Formen, welche metabolisch schwer zu generieren sind, [25b,27] scheint der helikale Koordinationsraum des Corrin-Liganden speziell entgegenzukommen.…”