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Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.
 
Creator Shahi, Puja
Kumar, Ish
Sharma, Ritu
Sanger, Shefali
Jolly, R S
 
Subject QR Microbiology
 
Description 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.
 
Publisher Wiley
 
Date 2006-06
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/150/1/jolly2006.pdf
Shahi, Puja and Kumar, Ish and Sharma, Ritu and Sanger, Shefali and Jolly, R S (2006) Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis. The FEBS journal, 273 (11). pp. 2374-87. ISSN 1742-464X
 
Relation http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2006.05244.x/pdf
http://crdd.osdd.net/open/150/