3D Structure Modeling of Alpha-Amino Acid Ester Hydrolase from Xanthomonas rubrilineans.

Alpha-amino acid ester hydrolase (EC 3.1.1.43, AEH) is a promising biocatalyst for the production of semi-synthetic ß-lactam antibiotics, penicillins and cephalosporins. The AEH gene from Xanthomonas rubrilineans (XrAEH) was recently cloned in this laboratory. The three-dimensional structure of XrAEH was simulated using the homology modeling method for rational design experiments. The analysis of the active site was performed, and its structure was specified. The key amino acid residues in the active site - the catalytic triad (Ser175, His341 and Asp308), oxyanion hole (Tyr83 and Tyr176), and carboxylate cluster (carboxylate groups of Asp209, Glu310 and Asp311) - were identified. It was shown that the optimal configuration of residues in the active site occurs with a negative net charge -1 in the carboxylate cluster. Docking of different substrates in the AEH active site was carried out, which allowed us to obtain structures of XrAEH complexes with the ampicillin, amoxicillin, cephalexin, D-phenylglycine, and 4-hydroxy-D-phenylglycine methyl ester. Modeling of XrAEH enzyme complexes with various substrates was used to show the structures for whose synthesis this enzyme will show the highest efficiency.

Fluorescent properties of firefly luciferases and their complexes with luciferin.

Fluorescence of luciferases from Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417, Trp-426) was studied. Analysis of quenching of tryptophan fluorescence showed that the tryptophan residue conserved in all luciferases is not accessible for charged quenchers, which is explained by the presence of positively and negatively charged amino acid residues in the close vicinity to it. An effective energy transfer from tryptophan to luciferin was observed during quenching of tryptophan fluorescence of both luciferases with luciferin. From the data on the energy transfer, the distance between the luciferin molecule and Trp-417 (419) in the luciferin luciferase complex was calculated: 11-15 A for P. pyralis and 12-17 A for L. mingrelica luciferases. The role of the conserved Trp residue in the catalysis is discussed.