EstA from Arthrobacter nitroguajacolicus Rü61a, a thermo- and solvent-tolerant carboxylesterase related to class C beta-lactamases.
The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Rü61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C beta-lactamases. The S-x-x-K motif that in beta-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50 degrees C to 60 degrees C. The enzyme was fairly thermostable. After 19 days at 50 degrees C and after 24 hours at 60 degrees C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.
Schütte
Marcus
M
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149, Münster.
Fetzner
Susanne
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010648:Phenylacetates / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010648:Phenylacetates; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria; D001618:beta-Lactamases; C025136:phenylacetic acid