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

Abstract

Long-chain acyl-CoA thioesterases (EC 3.1.2.2) hydrolyze acyl-CoA esters to nonesterified fatty acids and coenzyme A (CoASH) [1]. These are ubiquitously expressed in bacteria, yeast, plants and mammals, and in most cell compartments, such as endoplasmic reticulum, cytosol, mitochondria and peroxisomes. Several unrelated thioesterases have been purified to homogeneity from plants, animals, and bacteria, and the cDNAs encoding several of them have been cloned and sequenced [2][3][4][5][6][7]. Although the physiological functions of these enzymes remain largely unknown, it is speculated that they regulate lipid metabolism by maintaining appropriate concentrations of acyl-CoA, CoASH, and nonesterified fatty acids. The only established function for acyl-CoA thioesterases is in the termination of fatty acid synthesis in eukaryotes [8].Two thioesterases, I and II, that cleave acyl-CoA molecules in vitro have been characterized from 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 C 12 to C 18 chain length with V max and K m of 3.58-9.73 lmolAEmin )1 AE(mg protein) )1 and 2.66-4.11 lm, 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°C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70°C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C 2 to C 12 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°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.Abbreviations DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); TEM, transmission electron microscopy.

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

“…This group comprises hydrolases acting on thioester (thioesterase) and sulfate ester (sulfatase) bonds. Furthermore, thioesterases have also been shown to convert para -nitrophenol esters (Shahi et al, 2006 ; Kotowska et al, 2009 ). EstB1 shared 30% sequence identity with SdsA1 from Pseudomonas aeruginosa , which is a characterized alkyl-sulfatase (Hagelueken et al, 2006 ).…”

Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis

“…This product has a maximum absorbance at 412 nm ( A 412) with a molar absorption coefficient of 13.6 mM −1 cm −1 [ 46 ]. Acyl-CoA thioesterase activity of purified ptTES1 was measured spectrophotometrically by monitoring the increase in A 412 as previously described [ 19 , 47 ]. Briefly, each assay contained 50 mM KCl, 10 mM HEPES (pH7.5), 0.3 mM DTNB and appropriate amount of decanoyl-CoA.…”

“…The expression of acyl-CoA thioesterases has been detected in most cellular compartments, such as cytosol, endoplasmic reticulum, mitochondria and peroxisomes. Acyl-CoA thioesterases may regulate cellular lipid metabolism by controlling the composition and concentration of acyl-CoA, CoASH and free fatty acids, although their physiological functions remain largely unknown [ 19 ]. Based on the structural features and functional implication, acyl-CoA thioesterases include the α/β-fold hydrolase and the Hotdog-fold thioesterase/dehydratase subfamilies.…”

Formation of Amyloid Fibrils via Longitudinal Growth of Oligomers

“…Extraction and Purification of Thioesterase. Extraction and purification of thioesterase were carried out as described previously (16). Briefly, A. faecalis was grown at 37 °C to an absorbance at 600 nm (A 600 ) of 4.0 in a rich medium with peptone (1%) and beef extract (0.5%).…”

“…The fresh sample of purified thioesterase obtained by size exclusion chromatography over a Sephadex 4B column was concentrated by repeated ultrafiltration using a Centricon 50 kDa membrane, suspended in water at concentration of 600 µg/mL, and subjected to TEM. The electron micrograph showed the protein as granular micellelike structures having an average diameter of 21.6 nm (16). The protein formed very viscous solutions at a concentration of ∼600 µg/mL, and concentration beyond this level by ultrafiltration on a 50 kDa membrane was extremely slow.…”