title: Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica.
abstract: The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA.
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.forename: Anatoli
authors.initials: A
authors.lastname: Tchigvintsev
authors.forename: Pierre
authors.initials: P
authors.lastname: Petit
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.forename: Alexander U
authors.initials: AU
authors.lastname: Singer
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.forename: Elena
authors.initials: E
authors.lastname: Evdokimova
authors.forename: Olga
authors.initials: O
authors.lastname: Egorova
authors.forename: Claudio F
authors.initials: CF
authors.lastname: Gonzalez
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Chernikova
authors.forename: Michail M
authors.initials: MM
authors.lastname: Yakimov
authors.forename: Michael
authors.initials: M
authors.lastname: Kube
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism*; D000864:Antarctic Regions; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D044130:Oceanospirillaceae / Q000201:enzymology*; D009821:Oils / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D000838:Anions; D050505:Mutant Proteins; D009821:Oils; D043182:Carboxylesterase