Direct Genetic and Enzymatic Evidence for Oxidative Cyclization in Hygromycin B Biosynthesis

Abstract

Hygromycin B is an aminoglycoside antibiotic with a structurally distinctive orthoester linkage. Despite its long history of use in industry and in the laboratory, its biosynthesis remains poorly understood. We show here, by in-frame gene deletion in vivo and detailed enzyme characterization in vitro, that formation of the unique orthoester moiety is catalyzed by the α-ketoglutarate- and non-heme iron-dependent oxygenase HygX. In addition, we identify HygF as a glycosyltransferase adding UDP-hexose to 2-deoxystreptamine, HygM as a methyltransferase responsible for N-3 methylation, and HygK as an epimerase. These experimental results and bioinformatic analyses allow a detailed pathway for hygromycin B biosynthesis to be proposed, including the key oxidative cyclization reactions.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains

“…A set of conserved nonheme iron, α-ketoglutarate (Fe/AKG)-dependent oxygenases in the avilamycin and everninomicin gene clusters is proposed to be responsible for catalyzing formation of features requiring oxidation, specifically the orthoester linkages and a methylenedioxy bridge (Figure A, colored red and blue). This assignment of function is confirmed by the recent demonstration that HygX, the sole oxygenase implicated in hygromycin B biosynthesis, catalyzes the formation of the sole orthoester linkage of that natural product . Within avilamycin and everninomicin biosynthetic pathways, however, the assignment of Fe/AKG oxygenases to specific features has been complicated by the presence of multiple orthoester linkages and the highly decorated oligosaccharide scaffold.…”

“…This assignment of function is confirmed by the recent demon- stration that HygX, the sole oxygenase implicated in hygromycin B biosynthesis, catalyzes the formation of the sole orthoester linkage of that natural product. 10 Within avilamycin and everninomicin biosynthetic pathways, however, the assignment of Fe/AKG oxygenases to specific features has been complicated by the presence of multiple orthoester linkages and the highly decorated oligosaccharide scaffold. Three putative Fe/AKG oxygenases are encoded in each avilamycin and everninomicin gene cluster (avi, ava, eve, and evd).…”

Selective Transformations of Carbohydrates Inspired by Radical-Based Enzymatic Mechanisms

“…The ability to selectively “edit” a stereocenter allows organisms and chemists to generate vast stereochemical diversity quickly from widely available precursors. A number of methods to epimerize stereocenters on sugars have been identified enzymatically and implemented synthetically, most via reversible polar enolization mechanisms or oxidation–reduction sequences. − Enzymatically, polar isomerization mechanisms are thermodynamically controlled and deliver a dynamic mixture of starting material and product isomers . That is, while the regioselectivity of an enolization step may be templated by the active site of the enzyme, a thermodynamically controlled distribution of starting and product stereoisomers is ultimately obtained.…”

HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue

“…HygY is annotated as a twitch radical SAM enzyme encoded in the hyg gene cluster (GenBank accession number AJ628642.1), which is responsible for the biosynthesis of hygromycin B ( 1 ) in Streptomyces hygroscopicus (Figure ). − Hygromycin B is an orthosomycin aminoglycoside antibiotic notable for its spirocyclic ortho-δ-lactone moiety that links an aminoheptose derivative of galactose ( 4 ) with a pseudodissacharide derived from d -talose ( 3 ) and N -methyl-2-deoxystreptamine ( 2 ). , Formation of this linkage requires C2′-epimerization of a d -galactose residue to d -talose ( 3 ) catalyzed by HygY so as to facilitate later oxidative cylization catalyzed by the α-ketoglutarate dependent nonheme iron oxygenase HygX. − While HygY has been reported to catalyze the C2′-epimerization of the putative intermediate galacamine ( 7 ), the details of this reaction including a structural characterization of the product are unavailable. This report provides an initial investigation into the mechanism of HygY catalyzed epimerization and demonstrates that HygY is a latent SPASM/twitch dehydrogenase with this activity revealed upon mutation of a catalytic cysteine residue.…”

“…The spirocyclic ortho-δ-lactone linkage in 1 is highlighted in blue. Other tandem biosynthetic pathways are also possible especially with respect to 3- N -methylation catalyzed by the highly promiscuous SAM-dependent methyltransferase HygM. , …”