Nonprocessive [2 + 2]e ^- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases

Abstract

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive ½2 þ 2 e − reductions to release thioesterbound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e − reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.chain release | glycopeptidolipid | NAD(P)H | tyrosine-dependent oxidoreductase P olyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are multifunctional proteins that are known to produce a variety of complex natural products (1). While most of these natural products can be classified as secondary metabolites, in mycobacteria PKSs and NRPSs synthesize lipidic metabolites that are important for their survival and pathogenesis (2). The biosynthetic mechanism involves assembly-line repetitive condensation of specific monomeric units. During this process, the intermediates remain covalently tethered to the proteins through the thiol group of phosphopantetheine (ppant) moiety that is posttranslationally added onto the carrier domains (3). The ppantarm-bound substrate reaches out to the active centers of the various domains to facilitate successive catalytic steps. The chainreleasing domain then performs dual function of detaching the mature product and playing a role in determining the final structure of the metabolite.The most well-studied chain-releasing domains are thioesterases (TE) that hydrolyze the thioester bond to release linear as well as macrocyclic products (4). Recently, a new mechanism of chain release catalyzed by the reductase (R) domains has been identified. These domains utilize NAD(P)H as cofactor to reductively release the final product as aldehyde or alcohol (Fig. S1A) (5). Interestingly, 2 R domain homologues are shown to perform cofactor-independent Dieckmann's cyclization (referred to as R* domains) (6). Broadly, R domains show homology to the family of short-chain dehydrogenases/reductases (SDRs). The SDR family consists of tyrosine-dependent oxidoreductases that are known to share common sequ...

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

“…The isonitrile-modified fatty acyl chain is then condensed to both amino groups of Lys promoted by the NRPS (ScoA) and reductively released by the reduction (R) domain of the NRPS to form a terminal alcohol product INLP 2. This reductive release mechanism is consistent with the identified activity of the R domain in Rv0101, which contains a conserved Ser/ Tyr/Lys catalytic triad and was demonstrated to catalyze a fourelectron thioester reduction using the purified truncated R domain and a synthetic substrate (17). We propose that the extra acetyl moiety found in INLP 1 is due to the activity of a promiscuous acetyltransferase endogenous to E. coli for possible detoxification, and similar acetylation events have been observed in other shunt product biosynthesis (26).…”

“…3). Although ScoC seems to prefer a short-chain fatty acid substrate (such as crotonic acid as shown in INLP 1 and 2), MmaC likely favors a medium-chain fatty acid substrate (such as 2-decenoic acid) because previous biochemical and structural analyses of Rv0099, a homolog of MmaC from the same genus of Mycobacterium, showed that this AAL activated fatty acids of medium-chain length (16,17).…”

Structures of Mycobacterium tuberculosis FadD10 Protein Reveal a New Type of Adenylate-forming Enzyme

“…5C). This is consistent with the modeling study that shows a hexadecyl chain can be accommodated in the fatty acid binding tunnel of FadD10 and the study by Chhabraa et al (16) showing that FadD10 could utilize fatty acids with up to 16 carbons in the adenylation reaction. The lower product yield for hexadecanoic acid could reflect differences in solubility or affinity.…”

“…These findings suggest that M. tuberculosis FadD10 activates and transfers the fatty acyl chain to the cognate ACP-Rv0100. Chhabraa et al (16) recently showed that FadD10 does not acylate CoA but is able to transfer a radioactively labeled dodecanoyl moiety to Rv0100 in vitro. This suggests that FadD10 has FAAL activity even though it has a primary sequence more similar to FACLs.…”

Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids

“…Two of these ORFs correspond to non-ribosomal peptide synthetases, designated as mps1 and mps2. Each of these proteins possesses two sets of modules that can be postulated to be responsible for synthesizing the tetrapeptide backbone, and the novel C-terminal reductase domain present on non-ribosomal peptide synthetase has been recently shown to reductively release the chain as acyl-peptidyl-alaninol (17). However, the functional relevance of the polyketide synthase (PKS) protein cannot be predicted.…”