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Summary: The cell envelope of Mycobacterium tuberculosis (Mtb) possesses a repertoire of unusual lipids that are believed to play an important role in pathogenesis. In this review, we specifically focus on computational, biochemical and structural studies in lipid biosynthesis that have established functional role of polyketide synthases (PKSs) and fatty acyl-AMP ligases (FAALs). Mechanistic and structural studies with FAALs suggest that this group of proteins may have evolved from omnipresent fatty acyl-CoA ligases (FACLs). FAALs activate fatty acids as acyl-adenylates and transfer them on to the PKSs which then produce unusual acyl chains that are the components of mycobacterial lipids. FAALs are a newly discovered family of enzymes; whereas involvement of PKSs in lipid metabolism was not known prior to their discovery in Mtb. Since Mtb genome contains multiple homologs of FAALs and PKSs and owing to the conserved reaction mechanism and overlapping substrate specificity; there is tempting opportunity to develop ‘systemic drugs’ against these enzymes as anti-tuberculosis agents. [Copyright &y& Elsevier]

A novel iterative biosynthetic mechanism for multifunctional polyketide synthases reveals how the metabolic diversity of this enzyme family can arise by using existing scaffolds in novel combinations. [ABSTRACT FROM AUTHOR]

Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Architecturally at least three different types of PKSs have been discovered in the microbial world and recent years have revealed tremendous versatility of PKSs, both in terms of their structural and functional organization and in their ability to produce compounds other than typical secondary metabolites. Mycobacterium tuberculosis exploits polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its survival. The functional significance of the large repertoire of PKSs in Dictyostelium discoideum, perhaps in producing developmental regulating factors, is emerging. Recently determined structures of fatty acid synthases (FASs) and PKSs now provide an opportunity to delineate the mechanistic and structural basis of polyketide biosynthetic machinery. [Copyright &y& Elsevier]

Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Additional complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the molecular recognition features of polyketide synthases. Within this decade, a variety of genetic, biochemical, and chemical investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review. [ABSTRACT FROM AUTHOR]

Presents information pertaining to the modular polyketide synthases (PKSs). Identification of examples of the PKSs; Consistency of modules; How heterodimeric protein pairs were generated; Suggestion of results; What each polypeptide includes.

Studies the purification and characterization of bimodular and trimodular derivatives of the erythromycin polyketide synthases. Role of modules in single condensation and associated reduction reactions; Synthesis of the anticipated triketide lactone.

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 P212121, with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 Å. The crystal structure of the N-terminal domain of FadD28 at 2.35 Å resolution has been solved using the MAD method. [ABSTRACT FROM AUTHOR]

The termination module of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) offloads the final product as an acid (occasionally also accompanied by cyclization) upon hydrolysis by employing thioesterase domains (TE-domains). Reductase domains (R-domains) of short-chain dehydrogenase/reductase (SDR) family offer an alternative offloading mechanism by reducing 4′-phosphopantetheine (4′-PPant) arm-tethered peptidyl chain, a thioester, to an aldehyde or an alcohol. Recent studies have highlighted their functional importance, for instance in the glycopeptidolipid (GPL) biosynthesis of Mycobacterium smegmatis , where the resulting alcoholic group is the site for subsequent modifications such as glycosylations. The mechanistic understanding of how these R-domains function in the context of multi-modular NRPS and PKS is poorly understood. In this study, conformational differences in functionally important loops, not reported previously, were identified in a new crystal form of R-domain which may be relevant to functioning in the context of assembly-line NRPS and PKS enzymology. Here, we propose a concerted loop movement model that allows gating of cofactor binding to these enzymes, enabling the release of the final product only after the substrate has reached the active site during biosynthesis, and therefore distinct from a canonical single domain SDR family of enzymes. [ABSTRACT FROM AUTHOR]

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe- Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl- AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs. [ABSTRACT FROM AUTHOR]

Abstract: Activation of fatty acids as acyl-adenylates by fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis is a variant of a classical theme that involves formation of acyl-CoA (coenzyme A) by fatty acyl-CoA ligases (FACLs). Here, we show that FAALs and FACLs possess similar structural fold and substrate specificity determinants, and the key difference is the absence of a unique insertion sequence in FACL13 structure. A systematic analysis shows a conserved hydrophobic anchorage of the insertion motif across several FAALs. Strikingly, mutagenesis of two phenylalanine residues, which are part of the anchorage, to alanine converts FAAL32 to FACL32. This insertion-based in silico analysis suggests the presence of FAAL homologues in several other non-mycobacterial genomes including eukaryotes. The work presented here establishes an elegant mechanism wherein an insertion sequence drives the functional divergence of FAALs from canonical FACLs. [Copyright &y& Elsevier]

Abstract: Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis. [Copyright &y& Elsevier]

This article focuses on the precursors of polyketide biosynthesis. The study of bioactive natural products has undergone rapid advancement with the cloning and sequencing of large numbers of gene clusters. Polyketide synthases utilize acyl-coenzyme A (CoA) precursors and synthesize polyketides by repetitive decarboxylative condensations. The potential of these proteins to produce varied CoA precursors and their utilization in polyketide biosynthesis have not been investigated. Acyl-activating enzymes proteins provide an attractive route to expand the precursors available for polyketide biosynthesis.