CRYSTAL-STRUCTURE OF CARBOXYLESTERASE FROM PSEUDOMONAS-FLUORESCENS, AN ALPHA BETA HYDROLASE WITH BROAD SUBSTRATE-SPECIFICITY/

Background: A group of esterases, classified as carboxylesterases, hyd rolyze carboxylic ester bonds with relatively broad substrate specific ity and are useful for stereospecific synthesis and hydrolysis of este rs. One such carboxylesterase from Pseudomonas fluorescens is a homodi meric enzyme, consisting of 218-residue subunits. It shows a limited s equence similarity to some members of the alpha/beta hydrolase superfa mily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterase s is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate spe cificity of this carboxylesterase. Results: In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 Angstrom res olution. Each subunit consists of a central seven-stranded beta sheet flanked by six a helices, The structure reveals the catalytic triad as Ser114-His199-Asp168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 Angstrom, The inhibitor is covalently attached to Ser11 4 of both subunits, with the aromatic ring occupying a hydrophobic sit e defined by the aliphatic sidechains of Leu23, lle58, Ile70, Met73 an d Val170. No large structural changes are observed between the free an d inhibitor-bound structures. Conclusions: Carboxylesterase from P. fl uorescens has the alpha/beta hydrolase fold and the Ser-His-Asp cataly tic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interacti on to form a dimer; it may be these extra structural elements, not see n in other esterases, that account for the inability of carboxylestera se to hydrolyze long chain fatty acids. As a result of dimerization, t he active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the cataly tic residues are exposed to the solvent. An oxyanion hole, formed by n itrogen atoms of Leu23 and Gln115, is present in both the free and inh ibitor-bound structures. An open active site, as well as a large bindi ng pocket for the acid part of substrate, in P. fluorescens carboxyles terase may contribute to its relatively broad substrate specificity.