V. V. Fedorchuk

Pilot scale production and isolation of recombinant NAD+‐ and NADP+‐specific formate dehydrogenases

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.

Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae.

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+).

SITE-DIRECTED MUTAGENESIS OF THE FORMATE DEHYDROGENASE ACTIVE CENTRE : ROLE OF THE HIS332-GLN313 PAIR IN ENZYME CATALYSIS

Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of d‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H‐bond to the Gln313 carboxamide.

Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices

NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein α‐helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser‐131 and Ser‐160 were replaced by Ala, Val and Leu. Residues Ser‐168, Ser‐184 and Ser‐228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild‐type FDH. Double mutants S(131,160)A and S(184,228)A and the four‐point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild‐type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5–24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four‐point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild‐type enzyme.

Effect of interactions between amino acid residues 43 and 61 on thermal stability of bacterial formate dehydrogenases

NAD+-dependent formate dehydrogenases (EC 1.2.1.2, FDH) of methylotrophic bacteria Pseudomonas sp. 101 (PseFDH) and Mycobacterium vaccae N10 (MycFDH) exhibit high homology. They differ in two amino acid residues only among a total of 400, i.e., Ile35 and Glu61 in MycFDH substitute for Thr35 and Lys61 as in PseFDH. However, the rate constant for MycFDH thermal inactivation in the temperature range of 54-65°C is 4-6-times higher than the corresponding rate constant for the enzyme from Pseudomonas sp. 101. To clarify the role of these residues in FDH stability the dependence of the apparent rate constant for enzyme inactivation on phosphate concentration was studied. Kinetic and thermodynamic parameters for thermal inactivation were obtained for both recombinant wild-type and mutant forms, i.e., MycFDH Glu61Gln, Glu61Pro, Glu61Lys and PseFDH Lys61Arg. It has been shown that the lower stability of MycFDH compared to that of PseFDH is caused mainly by electrostatic repulsion between Asp43 and Glu61 residues. Replacement of Lys61 with an Arg residue in the PseFDH molecule does not result in an increase in stability.

Recombinant alpha-amino ester acid hydrolase from Xanthomonas rubrilineans VKPM B-9915 is a highly efficient biocatalyst of cephalexin synthesis

Recombinant, as well as native alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 (XrAEH, EC 3.1.1.43), was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that the recombinant enzyme r-XrAEH produced by Escherichia coli VKPM B-11246 is more efficient in comparison with the native enzyme wt-XrAEH prepared from mutant strain Xanthomonas rubrilineans VKPM B-9915. When r-XrAEH was used as a biocatalyst, addition of ethylene glycol (33 vol %) to the reaction medium improved the yield from 70 to 95%. During synthesis of cephalexin under optimal conditions in the case of the native enzyme wt-XrAEH the cephalexin yield was 85%, in contrast to r-XrAEH where it was 95%. Furthermore, unlike native wt-XrAEH enzymes, preparations of recombinant r-XrAEH do not possess beta-lactamase side activity.

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.

Alteration of the formate dehydrogenase isoelectric point by rational design

In order to extend the pH stability optimum for NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Pseudomonas sp. 101 (PseFDH), four mutant enzymes with Lys112Pro, Lys231Ala, Lys231Ser, and Lys317Asn substitutions were obtained by site-directed mutagenesis. The choice of the mutation sites and the types of substituting amino acids were based on the alignment of amino acid sequences of FDHs from various sources and the analysis of the three-dimensional structure of PseFDH. The kinetic properties and temperature stability were studied for all obtained mutant forms. It is shown that the substitutions in positions 112 and 231 slightly improved the kinetic properties; meanwhile, the Lys317Asn mutant possessed a decreased affinity for the coenzyme. A thermal stability assay for the obtained mutants revealed that the substitutions in positions 112 and 231 result in just a slight destabilization of the enzyme, while Lys317Asn substitution causes a significant decrease in thermal stability. The isoelectric point was decreased by 0.1 points for all obtained mutant forms.