A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases.

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.

Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results.

The optimisation of enzymes for particular purposes or conditions remains an important target in virtually all protein engineering endeavours. Here, we present a successful strategy for altering the pH-optimum of the triglyceride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment are correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we studied so far. This has prompted us to propose the "The Electrostatic Catapult Model" as a model for product release after cleavage of the ester bond. The origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutations into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge mutants displayed altered titration behaviour of active site electrostatic potentials. Typically, the substitution of the residue Glu44 pushes the onset of the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the experimentally observed. Finally, it was found that the pH-dependent computed Coulombic energy displayed a strong correlation with the observed melting temperatures of native cutinase.

Protein engineering the surface of enzymes.

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Crystallization and preliminary X-ray diffraction studies of the Pseudomonas marginata esterase EstB.

Crystals of the esterase EstB were obtained at 277 K with the hanging-drop technique from polyethylene glycol 4000 solutions containing 2-propanol at pH 7.5. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with cell dimensions a = b = 82.9 and c = 193.4 A (at 100 K). The crystals diffract beyond a resolution of 2.0 A.