Dioxane Bridge Formation during the Biosynthesis of Spectinomycin Involves a Twitch RadicalS-Adenosyl Methionine Dehydrogenase That May Have Evolved from an Epimerase

Abstract

Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2′ alcohol of (2′R,3′S)-tetrahydrospectinomycin to yield (3′S)dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2′ of the substrate by the 5′-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2′ of the bound substrate opposite C5′ of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2′ epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.

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 ).…”

Identification of the Early Steps in Herbicidin Biosynthesis Reveals an Atypical Mechanism of C-Glycosylation

“…Therefore, the B 12 -dependent radical SAM enzyme Her6 is proposed to catalyze the necessary epimerization of C3′–OH likely with 22 (with a 3′ S configuration) serving as the substrate. To date, no B 12 -dependent radical SAM enzyme has been reported to catalyze an epimerization reaction; however, radical SAM enzymes have been reported that do not depend on B 12 and are known to function as epimerases. − Subsequent cyclization to form ring B of herbicidin after C3′ epimerization need not be enzyme-catalyzed. The final step in the biosynthesis of 2 is then proposed to be the reduction of 33 , which is hypothesized to be catalyzed by Her7.…”

Crystal structure of BtrK, a decarboxylase involved in the (S)-4-amino-2-hydroxybutyrate (AHBA) formation during butirosin biosynthesis

“…Most aminoglycosides have a 2deoxystreptamine (2-DOS) moiety as an aglycone, with different amino-sugars attached to the carbons 4 and 5 or 4 and 6 such as in kanamycin, gentamicin, and neomycin [8]. In recent years, there were great advances in the understanding of biosyntheses and in the enzymology of several biosynthetic steps from aminoglycosides, particularly those 2-DOS-containing molecules [9][10][11][12][13][14][15][16].…”