Desferrioxamine biosynthesis: diverse hydroxamate assembly by substrate-tolerant acyl transferase DesC

Hydroxamate groups play key roles in the biological function of diverse natural products. Important examples include trichostatin A, which inhibits histone deacetylases via coordination of the active site zinc(II) ion with a hydroxamate group, and the desferrioxamines, which use three hydroxamate groups to chelate ferric iron. Desferrioxamine biosynthesis in Streptomyces species involves the DesD-catalysed condensation of various N -acylated derivatives of N -hydroxycadaverine with two molecules of N -succinyl- N -hydroxycadaverine to form a range of linear and macrocyclic tris-hydroxamates. However, the mechanism for assembly of the various N -acyl- N -hydroxycadaverine substrates of DesD from N -hydroxycadaverine has until now been unclear. Here we show that the desC gene of Streptomyces coelicolor encodes the acyl transferase responsible for this process. DesC catalyses the N -acylation of N -hydroxycadaverine with acetyl, succinyl and myristoyl-CoA, accounting for the diverse array of desferrioxamines produced by S. coelicolor . The X-ray crystal structure of DesE, the ferrioxamine lipoprotein receptor, in complex with ferrioxamine B (which is derived from two units of N -succinyl- N -hydroxycadaverine and one of N -acetyl- N -hydroxycadaverine) was also determined. This showed that the acetyl group of ferrioxamine B is solvent exposed, suggesting that the corresponding acyl group in longer chain congeners can protrude from the binding pocket, providing insights into their likely function. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology'. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

Structure ofN-myristoyltransferase fromAspergillus fumigatus

N-Myristoyltransferase (NMT) is an enzyme which translocates the 14-carbon saturated fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate peptides. This myristoylation process is involved in protein modification in various eukaryotes, including animals and fungi. Furthermore, this enzyme has been shown to be essential to the growth of various species, such asSaccharomyces cerevisiae, which indicates that NMT is an attractive target for the development of a novel antifungal drug. In this study, the crystal structure of a ternary complex of NMT fromAspergillus fumigatuswithS-(2-oxo)pentadecyl-CoA, a myristoyl-CoA analogue cofactor, and a synthetic inhibitor is reported at a resolution of 2.1 Å. The results advance the understanding of the specificity of NMT inhibitors and provide valuable information for structure-based drug design.

Diverse modes of binding in structures ofLeishmania majorN-myristoyltransferase with selective inhibitors

The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed.N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT fromLeishmania donovanihave been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely relatedL. majorare reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

Membrane microenvironment regulation of carnitine palmitoyltranferases I and II

CPT (carnitine palmitoyltransferase) 1 and CPT2 regulate fatty acid oxidation. Recombinant rat CPT2 was isolated from the soluble fractions of bacterial extracts and expressed in Escherichia coli. The acyl-CoA chain-length-specificity of the recombinant CPT2 was identical with that of the purified enzyme from rat liver mitochondrial inner membranes. The Km for carnitine for both the mitochondrial preparation and the recombinant enzyme was identical. In isolated mitochondrial outer membranes, cardiolipin (diphosphatidylglycerol) increased CPT1 activity 4-fold and the Km for carnitine 6-fold. It decreased the Ki for malonyl-CoA inhibition 60-fold, but had no effect on the apparent Km for myristoyl-CoA. Cardiolipin also activated recombinant CPT2 almost 4-fold, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylcholine activated the enzyme 3-, 2- and 2-fold respectively. Most of the recombinant CPT2 was found to have substantial interaction with cardiolipin. A model is proposed whereby cardiolipin may hold the fatty-acid-oxidizing enzymes in the active functional conformation between the mitochondrial inner and outer membranes in conjunction with the translocase and the acyl-CoA synthetase, thus combining all four enzymes into a functional unit.

Thioesterase superfamily member 2 (Them2)/acyl-CoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs

Them2 (thioesterase superfamily member 2) is a 140-amino-acid protein of unknown biological function that comprises a single hotdog fold thioesterase domain. On the basis of its putative association with mitochondria, accentuated expression in oxidative tissues and interaction with StarD2 (also known as phosphatidylcholine-transfer protein, PC-TP), a regulator of fatty acid metabolism, we explored whether Them2 functions as a physiologically relevant fatty acyl-CoA thioesterase. In solution, Them2 formed a stable homotetramer, which denatured in a single transition at 59.3 °C. Them2 exhibited thioesterase activity for medium- and long-chain acyl-CoAs, with Km values that decreased exponentially as a function of increasing acyl chain length. Steady-state kinetic parameters for Them2 were characteristic of long-chain mammalian acyl-CoA thioesterases, with minimal values of Km and maximal values of kcat/Km observed for myristoyl-CoA and palmitoyl-CoA. For these acyl-CoAs, substrate inhibition was observed when concentrations approached their critical micellar concentrations. The acyl-CoA thioesterase activity of Them2 was optimized at physiological temperature, ionic strength and pH. For both myristoyl-CoA and palmitoyl-CoA, the addition of StarD2 increased the kcat of Them2. Enzymatic activity was decreased by the addition of phosphatidic acid/phosphatidylcholine small unilamellar vesicles. Them2 expression, which was most pronounced in mouse heart, was associated with mitochondria and was induced by activation of PPARα (peroxisome-proliferator-activated receptor α). We conclude that, under biological conditions, Them2 probably functions as a homotetrameric long-chain acyl-CoA thioesterase. Accordingly, Them2 has been designated as the 13th member of the mammalian acyl-CoA thioesterase family, Acot13.