Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca.

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

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.

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.