Highly-Active Recombinant Formate Dehydrogenase from Pathogenic Bacterium Staphylococcus aureus: Preparation and Crystallization.

# These authors contributed equally to the work. NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

Effect of His6-tag Position on the Expression and Properties of Phenylacetone Monooxygenase from Thermobifida fusca.

Phenylacetone monooxygenase (EC 1.14.13.92, PAMО) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMО is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His6-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His6-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (kcat) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMО variants were in agreement within the published data for PAMO L (within the experimental error); however, the KM for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His6-tag.

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.

Stabilization of plant formate dehydrogenase by rational design.

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD+ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments - the differences in maxima of the melting curves (T(m)) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T(m) decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH - Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.

Bioinformatics-Structural Approach to the Search for New D-Amino Acid Oxidases.

D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S.

Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.

Bacteriolytic Activity Of Human Interleukin-2, Chicken Egg Lysozyme In The Presence Of Potential Effectors.

The bacteriolytic activity of interleukin-2 and chicken egg lysozyme in the presence of various substances has been studied. Glycine and lysine do not affect the activity of interleukin-2 but increase that of lysozyme, showing a bell-shape concentration dependence peaking at 1.5 mM glycine and 18 mM lysine. Arginine and glutamate activate both interleukin-2 and lysozyme with a concentration dependence of the saturation type. Aromatic amino acids have almost no effect on the activity of both interleukin-2 and lysozyme. Aromatic amines, tryptamine, and tyramine activate interleukin-2 but inhibit lysozyme. Peptide antibiotics affect interleukin and lysozyme similarly and exhibit maximum activity in the micromolar range of antibiotics. Taurine has no effect on the activity of interleukin-2 and lysozyme. Mildronate showed no influence on lysozyme, but it activated interleukin-2 with the activity maximum at 3 mM. EDTA activates both interleukin-2 and lysozyme at concentrations above 0.15 mM.

Human Interleukin-2 and Hen Egg White Lysozyme: Screening for Bacteriolytic Activity against Various Bacterial Cells.

The bacteriolytic activity of interleukin-2 and hen egg white lysozyme against 34 different species of microorganisms has been studied. It was found that 6 species of microorganisms are lysed in the presence of interleukin-2. All interleukin-2-sensitive microorganisms belong either to the Enterobacteriaceae, Bacillaceae, or the Lactobacillaceae family. It was also found that 12 species of microorganisms are lysed in the presence of lysozyme, and 16 species of microorganisms are lysed in the presence of sodium dodecyl sulfate (SDS). The bacteriolytic activity of interleukin-2 and lysozyme was studied at various pH values.

Improvement of the soy formate dehydrogenase properties by rational design.

Previous experiments on substitution of the residue Phe290 to Asp, Asn and Ser in NAD(+)-dependent formate dehydrogenase from soya Glycine max (SoyFDH) showed important role of the residue in enzyme thermal stability and catalytic properties (Alekseeva et al. Prot. Eng. Des. Sel., 2012a; 25: :781-88). In this work, we continued site-directed mutagenesis experiments of the Phe290 and the residue was changed to Ala, Thr, Tyr, Glu and Gln. All amino acid changes resulted in increase of catalytic constant from 2.9 to 3.5-4.7 s(-1). The substitution Phe290Ala led to KM (NAD+) decrease from 13.3 to 8.6 µM, and substitutions Phe290Tyr and Phe290Glu resulted in decrease and increase of KM (HCOO-) from 1.5 to 0.9 and -2.9 mM, respectively. The highest improvement of catalytic properties was observed for SoyFDH Phe290Ala which showed 2-fold higher catalytic efficiency with both substrates. Stability of mutants was examined by study of thermal inactivation kinetics and differential scanning calorimetry (DSC). All five amino acids provided increase of thermal stability of mutant SoyFDH in comparison with wild-type enzyme. Mutant SoyFDH Phe290Glu showed the highest improvement-the stabilization effect was 43 at 56°C. The DSC data agree with results of thermal inactivation kinetics. Substitutions Phe290Tyr, Phe290Thr, Phe290Gln and Phe290Glu provided Tm value increase 0.6°-6.6°. SoyFDH Phe290Glu and previously prepared SoyFDH Phe290Asp show similar thermal stability as enzymes from Candida boidinii and Mycobacterium vaccae N10 and have higher catalytic efficiency with NAD(+) compared with all described FDHs. Therefore, these mutants are very perspective enzymes for coenzyme regeneration in processes of chiral synthesis with dehydrogenases.

Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max.

Recently, we demonstrated that the amino acid substitutions Ala267Met and Ala267Met/Ile272Val (Alekseeva et al., Biochemistry, 2012), Phe290Asp, Phe290Asn and Phe290Ser (Alekseeva et al., Prot. Eng. Des. Select, 2012) in recombinant formate dehydrogenase from soya Glycine max (SoyFDH) lead to a significant (up to 30-100 times) increase in the thermal stability of the enzyme. The substitutions Phe290Asp, Phe290Asn and Phe290Ser were introduced into double mutant SoyFDH Ala267Met/Ile272Val by site-directed mutagenesis. Combinations of three substitutions did not lead to a noticeable change in the catalytic properties of the mutant enzymes. The stability of the resultant triple mutants was studied through thermal inactivation kinetics and differential scanning calorimetry. The thermal stability of the new mutant SoyFDHs was shown to be much higher than that of their precursors. The stability of the best mutant SoyFDH Ala267Met/Ile272Val/Phe290Asp turned out to be comparable to that of the most stable wild-type formate dehydrogenases from other sources. The results obtained with both methods indicate a great synergistic contribution of individual amino acid substitutions to the common stabilization effect.

The role of ala198 in the stability and coenzyme specificity of bacterial formate dehydrogenases.

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 KM (NAD+) value from 60 to 35 and from 80 to 45 µM for PseFDH and MorFDH, respectively, while the KM (HCOO-) 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(+).

Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices.

Hydrophobization of alpha-helices is one of the general approaches used for improving the thermal stability of enzymes. A total of 11 serine residues located in alpha-helices have been found based on multiple alignments of the amino acid sequences of D-amino acid oxidases from different organisms and the analysis of the 3D-structure of D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO, EC 1.4.3.3). As a result of further structural analysis, eight Ser residues in 67, 77, 78, 105, 270, 277, 335, and 336 positions have been selected to be substituted with Ala. S78A and S270A substitutions have resulted in dramatic destabilization of the enzyme. Mutant enzymes were inactivated during isolation from cells. Another six mutant TvDAAOs have been highly purified and their properties have been characterized. The amino acid substitutions S277A and S336A destabilized the protein globule. The thermal stabilities of TvDAAO S77A and TvDAAO S335A mutants were close to that of the wild-type enzyme, while S67A and S105A substitutions resulted in approximately 1.5- and 2.0-fold increases in the TvDAAO mutant thermal stability, respectively. Furthermore, the TvDAAO S105A mutant showed on average a 1.2- to 3.0-fold higher catalytic efficiency with D-Asn, D-Tyr, D-Phe, and D-Leu as compared to the wild-type enzyme.

NAD (+) -dependent Formate Dehydrogenase from Plants.

NAD(+)-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases.

Assessment of Formate Dehydrogenase Stress Stability In vivo using Inactivation by Hydrogen Peroxide.

Kinetic studies on hydrogen peroxide-induced inactivation of mutant formate dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of hydrogen peroxide, the decrease in enzyme activity follows first-order kinetics. Therefore, the first-order effective inactivation kinetic constants determined for various FDH forms at a constant H(2)O(2) concentration can be used as a quantitative measure of the enzyme stability. It was shown that two cysteine residues located in the active site formate- and coenzyme-binding domains (Cys145 and Cys255, respectively) make similar contributions to the enzyme stability, while the contribution of Cys354 is insignificant. The inactivation kinetics of wild-type PseFDH, mutant PseFDH Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium Staphylococcus aureus, higher plants Arabidopsis thaliana, and soya Glycine max, was studied. It was found that the stress-induced FDHs are at least 20 times more stable than the nonstress-induced PseFDH from Pseudomonas sp. 101 grown on methanol.

Protein engineering of penicillin acylase.

Penicillin acylases (PA) are widely used for the production of semi-synthetic ß-lactam antibiotics and chiral compounds. In this review, the latest achievements in the production of recombinant enzymes are discussed, as well as the results of PA type G protein engineering.

Influence of Ion Strength and pH on Thermal Stability of Yeast Formate Dehydrogenase.

The kinetics of the thermal inactivation of recombinant wild-type formate dehydrogenase from Candida boidinii yeast was studied in the temperature range of 53-61(o)C and pH 6.0, 7.0, and 8.0. It was shown that the loss of the enzyme's activity proceeds via a monomolecular mechanism. Activation parameters ∆Н(-) and ∆S(-) were calculated based on the temperature relations dependence of inactivation rate constants according to the transition state theory. Both parameters are in a range that corresponds to globular protein denaturation processes. Optimal conditions for the stability of the enzyme were high concentrations of the phosphate buffer or of the enzyme substrate sodium formate at pH = 7.0.