The atomic-resolution structure of a novel bacterial esterase

Background: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader Sequence of 27 amino acids suggests that this enzyme is exported from,the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology,to any known protein sequence. Results: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1.1 Angstrom resolution. The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230; The acidic residue of the catalytic triad (Glu230) is located on the beta 6 Strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta 7 strand. The oxyanion hole is formed by the: mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a-substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72. Conclusions: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison,with :Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of-the periplasmic space.