title: Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.
abstract: A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.
authors.affiliation: Institute of Microbial Technology, Chandigarh, India.
authors.forename: Puja
authors.initials: P
authors.lastname: Shahi
authors.forename: Ish
authors.initials: I
authors.lastname: Kumar
authors.forename: Ritu
authors.initials: R
authors.lastname: Sharma
authors.forename: Shefali
authors.initials: S
authors.lastname: Sanger
authors.forename: Ravinder S
authors.initials: RS
authors.lastname: Jolly
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D042481:Alcaligenes faecalis / Q000201:enzymology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D000102:Acetyl-CoA C-Acyltransferase