The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective to fatty acid activation dogma. These proteins convert fatty acids to corresponding adenylates, which is an intermediate of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens like Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyl-adenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of a FAAL protein and by generating loss- as well as gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyl-adenylate. Since FAALs in Mtb are crucial nodes in biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multi-pronged approach of simultaneously disrupting several pathways.

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...

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SummaryIntracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.

BackgroundLeishmania META1 has for long been a candidate molecule for involvement in virulence: META1 transcript and protein are up-regulated in metacyclic Leishmania. Yet, how META1 contributes to virulence remains unclear. We sought insights into the possible functions of META1 by studying its evolutionary origins.ResultsUsing multiple criteria including sequence similarity, nucleotide composition, phylogenetic analysis and selection pressure on gene sequence, we present evidence that META1 originated in trypanosomatids as a result of a lateral gene transfer of a bacterial heat-inducible protein, HslJ. Furthermore, within the Leishmania genome, META1 sequence is under negative selection pressure against change/substitution. Using homology modeling of Leishmania META1 based on solved NMR structure of HslJ, we show that META1 and HslJ share a similar structural fold. The best hit for other proteins with similar fold is MxiM, a protein involved in the type III secretion system in Shigella. The striking structural similarity shared by META1, HslJ and MxiM suggests a possibility of shared functions. Upon structural superposition with MxiM, we have observed a putative hydrophobic cavity in META1. Mutagenesis of select hydrophobic residues in this cavity affects the secretion of the secreted acid phosphatase (SAP), indicating META1's involvement in secretory processes in Leishmania.ConclusionsOverall, this work uses an evolutionary biology approach, 3D-modeling and site-directed mutagenesis to arrive at new insights into functions of Leishmania META1.

FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the first 460 residues, was crystallized by the hanging-drop vapour-diffusion method at 295 K. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 angstroms. The crystal structure of the N-terminal domain of FadD28 at 2.35 angstroms resolution has been solved using the MAD method.

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