It has been shown by an X-ray structural analysis that the amino acid residues
Ala198, which are located in the coenzyme-binding domain of
NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from
bacteria Pseudomonas sp.101 and Moraxella sp.
C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles
ψ and φ. These residues were replaced with Gly by site-directed
mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in
E.coli cells and obtained in active and soluble forms with
more than 95% purity. The study of thermal inactivation kinetics showed that
the mutation A198G results in a 2.5- fold increase in stability compared to one
for the wild-type enzymes. Kinetic experiments indicate that A198G replacement
reduces the KMNAD+ value from 60 to 35 and from 80 to 45
μM for PseFDH and MorFDH, respectively, while the
KMHCOO- value remains practically unchanged. Amino acid
replacement A198G was also added to the mutant PseFDH D221S with the coenzyme
specificity changed from NAD+ to NADP+. In this case, an
increase in thermal stability was also observed, but the influence of the
mutation on the kinetic parameters was opposite: KM increased from 190 to 280
μM and from 43 to 89 mM for NADP+ and formate, respectively.
According to the data obtained, inference could be drawn that earlier formate
dehydrogenase from bacterium Pseudomonas sp. 101 was specific
to NADP+, but not to NAD+.
d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.
Alpha-amino acid ester hydrolase (EC 3.1.1.43, AEH) is a promising biocatalyst
for the production of semi-synthetic β-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.
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