Crystallization and preliminary X-ray diffraction studies of the<i>Pseudomonas marginata</i>esterase EstB

Wagner, Ulrike; Sölkner, B.; Petersen, Evamaria; Schlacher, Anton; Schwab, Helmut; Kratky, C.

doi:10.1107/s0907444997004988

Cited by 2 publications

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“…The EstB esterase was expressed in E. coli , purified, and crystallized (hanging‐drop vapor diffusion from 0.1 M HEPES buffer pH 7.5, 10% 2‐propanol, and 20 % PEG 4000 at 4°C) to a final size of about 0.3 × 0.3 × 0.8 mm as described previously (Wagner et al 1997). Crystals belong to the trigonal space group P3 2 21 with cell dimensions of a = b = 83.4 and c = 194.6 Å.…”

Section: Methodsmentioning

confidence: 99%

“…In fact, when incubated with 7‐aminocephalosporinic acid (7‐ACA), which contains a β‐lactam ring and an ester group, it cleaves the ester bond but leaves the β‐lactam unaffected (Petersen et al 2001). Following the crystallization of this enzyme (Wagner et al 1997), we now report its crystal structure, which should yield insight into the structural factors discriminating between esterolytic and β‐lactam‐cleaving activity. To identify the active‐site nucleophile, and to probe possible conformational changes during catalysis, we also determined the structure of the reaction product between EstB and diisopropyl‐fluorophosphate (DFP).…”

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confidence: 98%

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EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

et al. 2002

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Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and ␤-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of ␤-lactamases and DD-peptidases. The chemical reactivities of esters and ␤-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying ␤-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C ␤-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "␤-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to ␤-lactamases, EstB shows no ␤-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous ␤-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus ␤-lactam cleavage.

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Section: Methodsmentioning

confidence: 99%

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confidence: 98%

EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

et al. 2002

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Enzymes, Directed Evolution

Reetz

2009

Encyclopedia of Industrial Biotechnology

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Directed evolution has emerged as a powerful method for engineering the catalytic profile of enzymes. It is based on repetitive cycles of random gene mutagenesis and expression coupled with efficient high‐throughput screening or selection for a given catalytic property such as thermostability, substrate acceptance, and/or enantioselectivity. In the 1990s, directed evolution was established on a broad front by applying standard mutagenesis methods such as error‐prone polymerase chain reaction (epPCR), saturation mutagenesis, and DNA shuffling. The current challenge is to devise optimal strategies for probing protein sequence space, thereby allowing for fast directed evolution. This article covers the progress of the last few years.

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Crystallization and preliminary X-ray diffraction studies of thePseudomonas marginataesterase EstB

Cited by 2 publications

References 10 publications

EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

EstB from Burkholderia gladioli: A novel esterase with a β‐lactamase fold reveals steric factors to discriminate between esterolytic and β‐lactam cleaving activity

Enzymes, Directed Evolution

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