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...

Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture.

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Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ-/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.