Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates

Abstract

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,β-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF--an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C-C bond formation not previously observed in nature, to create an 11-carbon chain.

Quinovosamycins: new tunicamycin-type antibiotics in which the α, β-1″,11′-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine

“…13,14 The catalytic activity of TunB is to generate the 11-carbon sugar tunicamine from 6-carbon 5′,6′-exo-glycal intermediates which are themselves generated from UDP-GlcNAc. 20 Using the tunB and tunD gene sequences as probes of an actinomycetes genomic library we identified seven microorganisms with the potential for TUN biosynthesis, four of which are previously unreported. These include Streptomyces sp.…”

Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3′‐Deoxy Nucleoside of Pacidamycins

“…All dehydratases studied so far that are involved in carbohydrate processing, to the best of our knowledge, have been shown to be large, multimeric and in general to require co‐factors (commonly metal ions or nucleotide) or needing covalent interactions for catalysis. A handful of dehydratases studied so far utilise a more unusual E1cb mechanism, these include the well‐studied dTDP‐ d ‐glucose 4,6‐dehydratase RmlB6a and the UDP‐Glc N Ac 5,6‐dehydratase TunA,6b both nicotinamide‐dependent (Figures S15 and S16, SI p. 21). RmlB and TunB mediate an NAD + assisted oxidation of a hydroxy group within their nucleotide substrates, followed by the enzymes’ mediation of an E1cb dehydration to generate the respective enones, and a subsequent reduction of the resultant conjugated C=C double bond in the case of RmlB (Figure S16) 6a.…”

Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis*

“…Our kinetics data (k cat/ K m ) for Pen suggest that it converts UDP-GlcNAc into product more efficiently (Table 3) compared with TunA and PseB (27,38). However, the product itself inhibited the Pen forward reaction.…”

“…It is not known whether the UDP-GlcNAc derivative participates in other metabolic pathways. Interestingly, UDP-6-deoxy-D-GlcNAc-5,6-ene is an intermediate formed by TunA during the synthesis of tunicamycin by Streptomyces species (38). Whether or not the pathway of an analogue exists in B. thuringiensis remains unknown.…”

“…Whether or not the pathway of an analogue exists in B. thuringiensis remains unknown. Although the TunA enzyme structure was solved (38), very low amino acid sequence similarity (24% and e-value 0.004) exists between the Pen and the TunA protein, suggesting these genes are evolutionarily not related and perhaps arose from different ancestral genes. With regard to Pal, we are currently unaware of another single activity identical to this enzyme.…”