Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs.

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.

Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta).

Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17beta-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (K(i)= approximately 9microM) and is a weaker, noncompetitive inhibitor of AEAT (K(i)= approximately 31microM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17beta-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17beta-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis-the fatty acid esterification of testosterone.

The adsorption of substrate-binding domain of PHB depolymerases to the surface of poly(3-hydroxybutyric acid).

The binding characteristic of PHB depolymerase has been studied by using glutathione S-transferase (GST) fusion proteins with substrate-binding domain of three bacterial PHB depolymerases, Alcaligenes faecalis, Comamonas acidovorans and Comamonas testosteroni. Analysis using immuno-gold labeling technique and transmission electron microscopy indicated that a novel GST fusion protein derived from A. Faecalis enzyme adsorbed to the surface of poly(3-hydroxybutyric acid) (P(3HB)) single crystals like other fusion proteins. Comparison of inhibiting degree of P(3HB) powder hydrolysis activity of PHB depolymerase by fusion proteins indicated that three fusion proteins bind to P(3HB) powder in the same degree. The measurement of the surface hydrophobicity of proteins suggests that the interaction of the substrate-binding domain with insoluble P(3HB) may include not only a hydrophobic effect but also molecule-specific contacts.

Total synthesis and in vitro-antifungal activity of (+/-)-2-methoxytetradecanoic Acid.

The marine fatty acid (+/-)-2-methoxytetradecanoic acid was synthesized in two steps (71% overall yield) starting from commercially available methyl-2-hydroxy-tetradecanoate. The title compound was antifungal against Candida albicans (ATCC 14053) in RPMI medium and Aspergillus niger (ATCC 16404) and Cryptococcus neoformans (ATCC 66031) in SDB medium at the minimum inhibitory concentration (MIC) of 100 mM, which compares to the fungitoxicity of a 2-iodotetradecanoic acid against the same fungi. The title compound was also five to ten times more cytotoxic than capric acid to C. albicans and A. niger in the tested medium but comparable in cytotoxicity to either capric acid and its 2-methoxylated analog to C. neoformans. The antifungal activity of (+/-)-2-methoxytetradecanoic acid is explained in terms of inhibition of N-myristoyltransferase.