S. S. Savin

Role of a Structurally Equivalent Phenylalanine Residue in Catalysis and Thermal Stability of Formate Dehydrogenases from Different Sources.

Comparison of amino acid sequences of NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2) from different sources and analysis of structures of holo-forms of FDH from bacterium Pseudomonas sp. 101 (PseFDH) and soya Glycine max (SoyFDH) as well as of structure of apo-form of FDH from yeast Candida boidinii (CboFDH) revealed the presence on the surface of protein globule of hydrophobic Phe residue in structurally equivalent position (SEP). The residue is placed in the coenzyme-binding domain and protects bound NAD+ from solvent. The effects of amino acid changes of the SEP on catalytic properties and thermal stability of PseFDH, SoyFDH, and CboFDH were compared. The strongest effect was observed for SoyFDH. All eight amino acid replacements resulted in increase in thermal stability, and in seven cases, increase in catalytic constant was achieved. Thermal stability of SoyFDH after mutations Phe290Asp and Phe290Glu increased 66- and 55-fold, respectively. KM values of the enzyme for substrate and coenzyme in different cases slightly increased or decreased. In case of one CboFDH, the mutein catalytic constant increased and thermal stability did not changed. In case of the second CboFDH mutant, results were the opposite. In one PseFDH mutant, amino acid change did not influence the catalytic constant, but in three others, the parameter was reduced. Two PseFDH mutants had higher and two mutants lower thermal stability in comparison with initial enzyme. Analysis of results of SEP mutagenesis in FDHs from bacterium, yeast, and plant shows that protein structure plays a key role for effect of the same amino acid changes in equivalent position in protein globule of formate dehydrogenases from different sources.

Influence of Met/Leu amino acid changes on catalytic properties and oxidative and thermal stability of yeast D-amino acid oxidase

The oxidative stability of enzymes is mostly dependent on the stability of the Cys and Met residues. Three single point mutants with Met/Leu substitutions in D-amino acid oxidase (DAAO, EC 1.4.3.3) from the yeast Trigonopsis variabilis (TvDAAO) are prepared and characterized. The positions for the amino acid residue substitutions are selected based on multiple alignment of different DAAO amino acid sequences and analysis of the three-dimensional structure of TvDAAO. It is shown that the substrate specificity profile ischanged for all of the mutants. The KM values for the small and bulky D-amino acidsare increased and decreased, respectively. One of the Met/Leu substitutions results in a two- to threefold increase in thermal stability as compared to the wild-type enzyme. A method for the determination of TvDAAO stability in the presence of hydrogen peroxide is developed and the oxidative stability of wild-type and mutant TvDAAOs is studied. It is shown that none of thethree mutations changes the oxidative stability of the enzymes.

Preparation and characterization of multipoint yeast D-amino acid oxidase mutants with improved stability and activity

D-amino acid oxidase (DAAO) is an FAD-containing oxidoreductase that stereospecifically oxidases D-amino acids to produce α-keto-acids, an ammonium ion, and hydrogen peroxide. The most important biotechnological process involving DAAO is the production of 7-amino cephalospranic acid (7-ACA) from cephalosporin C. The reaction product, 7-ACA, is then used as a precursor for the synthesis of cephalosporin antibiotics of different generations. We previously obtained mutant DAAOs from the yeast Trigonopsis variabilis (TvDAAO). The mutants with point amino acid substitutions were characterized by either an increased thermal stability or improved catalytic properties in the oxidation of cephalosporin C. In the present study, we obtained two new mutant TvDAAOs with two and four amino acid substitutions, respectively. The catalytic constants of these mutant TvDAAOs for the oxidation of cephalosporin C were 1.8 and 4 times higher than the respective parameter of the wild-type enzyme (wt-TvDAAO). The combination of substitutions increased the thermal stabilities of both mutant TvDAAOs by a factor of 2–3 as compared with the wt- TvDAAO.

Comparison of bacteriolytic activity of human interleukin-2 and chicken egg lysozyme on Lactobacillus plantarum and Escherichia coli cells

The article continues studies of the recently discovered bacteriolytic activity of interleukin-2. It was detected earlier that interleukin (IL-2) possesses greater substrate specificity in comparison with chicken egg lysozyme. IL-2 disrupted the cell wall of Escherichia coli but did not lyse lysozyme substrates such as the cell walls of Micrococcus luteus and Bacillus subtilis. In the present study it is demonstrated for the first time that both IL-2 and chicken egg lysozyme are capable of lysing Lactobacillus plantarum. The effects of IL-2 and chicken egg lysozyme on Lactobacillus plantarum are compared with those on Escherichia coli. The dependences of the rate of lysis on the concentration of bacteriolytic factors and pH are studied.

Membrane detection of nanogram amounts of formate dehydrogenase

A method for the detection of nanogram amounts of formate dehydrogenase on membranes was developed. The optimization of the method was carried out for determination of the best mediator concentration and tetranitrotetrazolium blue. It was found that 5-methyl-phenazil methosulfate is a more effective mediator than phenazine methosulfate. Different types of membranes were tested and it was shown that nitrocellulose membrane provides a low background and the highest sensitivity. Under optimal conditions the detection limit was 15 ng of the enzyme in the probe.

Inactivation of formate dehydrogenase at pH 8

The first-order inactivation rate constant as a function of the phosphate buffer concentration has been studied for recombinant formate dehydrogenases from plants Arabidopsis thaliana and soybean and for mutant formate dehydrogenase from bacterium Pseudomonas sp. 101 (PseFDH GAV). Both stabilization and destabilization of the enzyme can be observed depending on the ionic strength of the buffer.

Enzymatic Lysis of Living Microbial Cells: A Universal Approach to Calculating the Rate of Cell Lysis in Turbidimetric Measurements

The data of the turbidimetric measurement for the enzymatic lysis of various living bacterial cells are analyzed. A method for the correct recalculation of the turbidimetric data (–ΔA/Δt) into absolute values of the change in the concentration of living cells (–ΔCFU/Δt) is proposed. The dimensionless efficiency of cell lysis–(1/CFU0) · ΔCFU/Δt for various bacterial cells is calculated to correctly compare the efficiency of the action of different bacteriolytic factors on various bacterial cells.

Rational Design of Practically Important Enzymes

Majority of native enzymes are poorly applicable for practical usage: that is why different methods of enzyme modification are used to obtain the biocatalysts with appropriate characteristics. Development of genome sequencing and various modern approaches in protein engineering allow one to identify protein of interest and to improve the enzyme properties for a particular process. This review describes the results on development of novel biocatalysts based on bioinformatics and rational design. New genes encoding formate dehydrogenase (FDH) from bacterium Staphylococcus aureus, yeasts Ogataea parapolymorpha and Saccharomyces cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH and PpaFDH, respectively), have been cloned. New FDHs were produced in the active form and characterized. SauFDH was shown to have at least 2-fold higher catalytic constant than other known FDHs. OpaFDH has catalytic parameters as good as those for soy FDH mutant forms, and in addition, is more thermostable. Apo- and holo-forms of SauFDH have been crystallized. Mutation of two Cys residues in Pseudomonas sp.101 enzyme (PseFDH) yields enzyme preparations with improved kinetic parameters and enhanced thermal and chemical stability. New generation of PseFDH preparations with the coenzyme specificity changed from NAD+ to NADP+ have been obtained. The effect of ionic liquids on the catalytic properties and thermal stability of six wild-type recombinant FDHs, and a number of their mutants, have been studied. In case of D-amino acid oxidase (DAAO), single-point mutations have been combined to create multi-point mutants. The introduced amino acid replacements have been shown to exert an additive effect, improving both kinetic parameters and increasing thermal and chemical stability. DAAO genes from Hansenula polymorpha yeast have been cloned. α-Amino acid ester hydrolase (AEH) gene has been cloned and expressed in the active form in E. coli. Structural modeling has been performed and the effectiveness in amino beta-lactams synthesis studied. The structure of a single-chain penicillin acylase from Alcaligenes faecalis (scAfPA) has been modeled and two variants of scAfPA gene was generated by PCR. Both variants have been expressed in E. coli, isolated and characterized. Catalytic properties of scAfPA were slightly better than those of its natural heterodimer.

Comparison of Thermal Stability of New Formate Dehydrogenases by Differential Scanning Calorimetry

Formate dehydrogenases (FDHs) from different sources are systematically studied in our laboratory. Over the past few years, new genes of four FDHs from pathogenic bacterium Staphylococcus aureus (SauFDH), methylotrophic thermotolerant yeast Ogataea parapolymorpha (OpaFDH), yeast Saccharomyces cerevisiae (SceFDH), and moss Physcomitrella patens (PpaFDH) have been cloned and expressed in E. coli cells. By means of differential scanning calorimetry, a comparative study of thermal stability of new recombinant formate dehydrogenases and a number of FDHs from other sources has been performed. It was shown that two new enzymes, SauFDH and OpaFDH, are comparable to the FDH from Pseudomonas sp. 101 bacteria in their stability. SceFDH is the least stable FDH among the described formate dehydrogenases.

The 3D-structural modeling of yeast D-amino acid oxidase

D-amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is used in the pharmaceutical industry and fine organic synthesis but for future practical applications new mutant forms of the enzyme with improved stability and catalytic properties are needed. Experiments on the crystallization of TvDAAO have been carried out for the last three decades without any success. For protein engineering of the enzyme using a rational design approach a model 3D-structure of TvDAAO was built using the homology modeling method. Cys108 and Cys298 residues were proposed for site-directed mutagenesis after analysis of the enzyme model structure.