A bacterial esterase is homologous with nonhaem haloperoxidases and displays brominating activity

Abstract

Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.

Catalytic Promiscuity in Biocatalysis: Using Old Enzymes to Form New Bonds and Follow New Pathways

“…For example, esterase from Pseudomonas fluorescens, [36] a lactonase, [37] and many lipases, [38] show low peroxidase activity in the presence of a carboxylic acid and hydrogen peroxide. Conversely, at least one haloperoxidase shows low esterase activity.…”

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

“…[11,49] bAChE and hAChE were expressed in Pichia pastoris as published and kindly provided as crude, non-lyophilized extracts by S. Vorlová for investigation. [50] Cell transformation, grows and protein expression:…”

Integrating Structure, Bioinformatics, and Enzymology to Discover Function

“…Enzyme Assays-Rapid screening for enzyme activities were performed using the following procedures: (a) fatty acid esterase activity was measured spectrophotometrically at 37°C using p-nitrophenyl (pNP) acetate or pNP esters of other fatty acids (C3-C18) as substrates (5), (b) thioesterase activity was measured spectrophotometrically using CoA thioesters of fatty acids (acetyl-CoA, malonyl-CoA, and palmitoyl-CoA) as described earlier (6), (c) lipase activity (with sonicated olive oil as substrate) was measured spectrophotometrically by the copper soap assay after extraction of released free fatty acids with chloroform: heptane:methanol mixture (7), (d) protease activity was measured using L-leucine p-nitroanilide (aminopeptidase activity) or N␣-benzoyl-Larginine p-nitroanilide (trypsin-like endopeptidase activity) as described (8,9), (e) phosphatase activity was determined spectrophotometrically using 5 mM p-nitrophenyl phosphate in 50 mM HEPES-K (pH 7.5) buffer at 37°C (10), and (f) bromoperoxidase activity was measured spectrophotometrically with phenol red or monochlorodimedon as described previously (11).…”