The soluble acyl-acyl carrier protein synthetase of Vibrio harveyi B392 is a member of the medium chain acyl-CoA synthetase family.

The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.

Crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of FadD28, a fatty-acyl AMP ligase from Mycobacterium tuberculosis.

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 P2(1)2(1)2(1), with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 angstroms. The crystal structure of the N-terminal domain of FadD28 at 2.35 angstroms resolution has been solved using the MAD method.

A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy.

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis.

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster.

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the alpha subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.