title: Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols.
abstract: An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Rehdorf
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Behrens
authors.forename: Giang-Son
authors.initials: GS
authors.lastname: Nguyen
authors.forename: Robert
authors.initials: R
authors.lastname: Kourist
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000438:Alcohols / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D004950:Esterases