Search

Summary Mycobacterium tuberculosis ( Mtb ) can survive in hypoxic necrotic tissue by assimilating energy from host-derived fatty acids. While the expanded repertoire of β-oxidation auxiliary enzymes is considered crucial for Mtb adaptability, delineating their functional relevance has been challenging. Here, we show that the Mtb fatty acid degradation (FadAB) complex cannot selectively break down cis fatty acyl substrates. We demonstrate that the stereoselective binding of fatty acyl substrates in the Mtb FadB pocket is due to the steric hindrance from Phe287 residue. By developing a functional screen, we classify the family of Mtb Ech proteins as monofunctional or bifunctional enzymes, three of which complement the FadAB complex to degrade cis fatty acids. Crystal structure determination of two cis - trans enoyl coenzyme A (CoA) isomerases reveals distinct placement of active-site residue in Ech enzymes. Our studies thus reveal versatility of Mtb lipid-remodeling enzymes and identify an essential role of stand-alone cis - trans enoyl CoA isomerases in mycobacterial biology. [ABSTRACT FROM AUTHOR]

Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order ‘bi–bi’ reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4′-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (R NRP ) and Mycobacterium smegmatis (R GPL ) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi–bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3–4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation–π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi–bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology. [ABSTRACT FROM AUTHOR]

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 [2 + 2]e- reductions to release thioester-bound 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. [ABSTRACT FROM AUTHOR]

Polyketide synthases (PKSs) are multi-functional enzymes that exhibit a great deal of diversity in their biosynthetic mechanism. This versatility allows them to influence an organism's biology in varied ways. Although many polyketides have been identified till now, and their biosynthetic pathways characterized, many more are waiting to be uncovered. With genome sequences of many organisms available now, the repository of putative pks genes is growing and so is the complexity involved in determining their possible products and functional roles in nature. Needless to say, efforts at the identification of novel products and pathways require a systematic, multi-pronged approach. In this review we discuss the growth of the PKS field and how it has undergone a pronounced transformation. We examine the various ways by which nature introduces variations in the products formed by these enzymes in different organisms to suit their biology. We further examine the different tools employed to crack the 'PKS puzzle' and how the future looks for research in this field. [ABSTRACT FROM AUTHOR]

The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalently sequestered biosynthetic intermediates are transferred from one enzymatic complex to another. Here we show that some of the 36 annotated fadD genes, located adjacent to the PKS genes in the Mycobacterium tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP ligases (FAALs). These proteins activate long-chain fatty acids as acyl-adenylates, which are then transferred to the multifunctional PKSs for further chain extension. This mode of activation and transfer of fatty acids is contrary to the previously described universal mechanism involving the formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in the biosynthesis of other lipid-containing metabolites and could have implications in engineering novel hybrid products. [ABSTRACT FROM AUTHOR]