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...
Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.
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