👤 David E Cane

Single nucleotide polymorphisms (SNPs) close to the VPS13C, C2CD4A and C2CD4B genes on chromosome 15q are associated with impaired fasting glucose and increased risk of type 2 diabetes. eQTL analysis revealed an association between possession of risk (C) alleles at a previously implicated causal SNP, rs7163757, and lowered VPS13C and C2CD4A levels in islets from female (n = 40, P < 0.041) but not from male subjects. Explored using promoter-reporter assays in β-cells and other cell lines, the risk variant at rs7163757 lowered enhancer activity. Mice deleted for Vps13c selectively in the β-cell were generated by crossing animals bearing a floxed allele at exon 1 to mice expressing Cre recombinase under Ins1 promoter control (Ins1Cre). Whereas Vps13c(fl/fl):Ins1Cre (βVps13cKO) mice displayed normal weight gain compared with control littermates, deletion of Vps13c had little effect on glucose tolerance. Pancreatic histology revealed no significant change in β-cell mass in KO mice vs. controls, and glucose-stimulated insulin secretion from isolated islets was not altered in vitro between control and βVps13cKO mice. However, a tendency was observed in female null mice for lower insulin levels and β-cell function (HOMA-B) in vivo. Furthermore, glucose-stimulated increases in intracellular free Ca(2+) were significantly increased in islets from female KO mice, suggesting impaired Ca(2+) sensitivity of the secretory machinery. The present data thus provide evidence for a limited role for changes in VPS13C expression in conferring altered disease risk at this locus, particularly in females, and suggest that C2CD4A may also be involved. Show less

Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water. Show less

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions. Show less

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multienzymes are organized into a series of functional units known as modules. Each dimeric module contains two catalytically independent clusters of active sites homologous to those of vertebrate fatty acid synthases. Earlier studies have shown that modules consist of head-to-tail homodimers in which ketosynthase (KS) and acyl carrier protein (ACP) domains are contributed by opposite subunits to form a catalytic center. Here, we probe the functional topology of the acyltransferase (AT) domain which transfers the methylmalonyl moiety of methylmalonyl-CoA onto the phosphopantetheine arm of the ACP domain. Using a bimodular derivative of DEBS, the AT domain of module 2 (AT2) was inactivated by site-directed mutagenesis. Heterodimeric protein pairs were generated in vitro between the inactivated AT2 (AT2 degrees) polypeptide and an inactive KS1 (KS1 degrees) or KS2 (KS2 degrees) protein. Both of these hybrid proteins supported polyketide synthesis, suggesting that AT2 can perform its function from either subunit. The apparent catalytic rate constants for each of the two hybrid protein pairs, KS1 degrees/AT2 degrees and KS2 degrees/AT2 degrees, were identical, indicating that no significant kinetic preference exists for a particular AT2-ACP2 combination. These results suggest that the AT domain can be shared between the two clusters of active sites within the same dimeric module. Such a novel structural organization might provide a functional advantage for the efficient biosynthesis of polyketides. Show less

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs. Show less