FDH

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Introduction

NAD⁺-dependent formate dehydrogenase (EC 1.2.1.2, FDH) is responsible for catalyzing the oxidation of formate ion to carbon dioxide and at the same time reducing NAD⁺ to NADH. The enzyme was first discovered in pea seeds, and intensive research was conducted in the 1970s, mainly for the formate dehydrogenase from methylotrophic bacteria and yeast. FDH belongs to the superfamily of D-specific dehydrogenases of 2-hydroxy acids, which is characterized by its active center having a similar structure and almost the same catalytically essential amino acid residues. Since this enzyme catalyzes the simplest reaction among other enzymes in the superfamily devoid of any proton release or abstraction steps, FDH is often chosen as a model enzyme.  

In the past ten years, FDH genes have been discovered in various organisms, including pathogens such as Staphylococcus aureus, Mycobacterium avium subsp. paratuberculosis str.k10, different strains of Bordetella and Legionella, Histoplasma capsulatum, Cryptococcus neoformans var. neoformans JEC21, etc. A large number of studies have shown that under certain conditions, FDH can play a key role in cell function. For example, FDH seems to be a stress protein in plants. It localizes to mitochondria, and its biosynthesis increases dramatically under stress conditions, up to 9% of the total mitochondrial protein. The expression of FDH gene is also stage-specific in fungal pathogens.

The general scheme of NAD(P)H regeneration for cofactor coupling enzymatic synthesis of optically active compounds is shown in the figure. The main enzyme E_p (dehydrogenase, reductase, monooxygenase, etc.) uses reduced cofactors to catalyze the production of chiral compounds, while the second enzyme E_R (such as formate dehydrogenase) reduces oxidized coenzymes to NAD(P)H. Many evidences indicate that FDH is one of the best enzymes for reduced cofactor regeneration. The reaction catalyzed by FDH fits all the criteria for NAD(P)H regeneration.

Figure 1. General scheme of NAD(P)H regeneration for cofactor coupled enzymatic synthesis of optically active compounds

Structure analysis applied to FDH engineering

X-ray data analysis was used to select the mutation positions for FDH from Pseudomonas sp.101 and the highly homologous (different only two amino acid residues) FDH from Mycobacterium vaccae N10. In 1993, high-resolution structures of apo-PseFDH (2NAC) and ternary complex (PseFDH NAD⁺-azide) (2NAD) were obtained. The analysis of PseFDH structure in complex with formate, ADP-ribose, NADH and (NADH + formate) revealed their intermediate feature between the 2NAC and 2NAD structures, i.e., apo-enzyme transformation into a holo-enzyme. All complexes are obtained with native enzyme purified from Pseudomonas sp.101. The presence of seven additional amino acid residues at the C-terminus of the recombinant wt-PseFDH protein interferes with crystallization. Recombinant FDH crystals were successfully obtained by removing these residues by mutagenesis. The full-size 400 aa polypeptide crystals have produced two mutant forms, namely PseFDHGAV and PseFDH T7, which have improved thermal stability.

Scientists have failed to resolve the structure data of wild-type CboFDH after many attempts. In order to obtain the required CboFDH crystals, a method based on the introduction of amino acid replacements in the highly disordered regions has been applied. Use special programs and the structure of homologous enzymes to predict new enzymes in these regions. In the case of CboFDH, this method is used to introduce the following mutations: Lys47Val, Lys47Glu, Arg296Ala, Lys328Val and Lys338Ala. Replacement Lys47Glu in CboFDH successfully prepared high-quality crystals, and finally obtained an apo-enzyme structure with a resolution of 1.9 Å. It is worth noting that Lys47 (Lys75 in PseFDH) is conserved in all 51 FDH complete and partial sequences known so far.

Enhancement of catalytic activity of FDH from C. boidinii

One of the disadvantages of FDH is its low catalytic activity. According to reports, the specific activity of bacterial enzymes is relatively high. At 30 °C, the specific activity of PseFDH is ca 10 U per mg of protein. Enzymes from other sources are not as active as bacterial FDH, and the activity of CboFDH is ca. 6.1-6.3 U/mg (30 °C). However, due to the different molecular mass of these enzymes (44,000 and 40,370 Da for bacterial and yeast enzymes, respectively), the values of k_cat of PseFDH and CboFDH are very different, 7.3 and 3.7 s^-1 respectively. Among the clones generated by random mutation of CboFDH, clones with a higher enzyme specific activity than wild-type CboFDH have been identified. Based on this result, scientists have produced CboFDH Phe285Tyr. The mutation does not affect the enzyme activity but increases its thermal stability. The Phe285 (311 PseFDH) residue in FDH is in - 2 position with respect to the catalytically important Gln287 (313 PseFDH) residue, which is located at the entrance of the enzyme active center at the site of the substrate binding channel. The increase in CboFDH activity caused by the replacement of Phe285Ser by up to 9.1 U/mg will not affect the thermal stability of the enzyme, but slightly worsened the K_m value of coenzyme and formate.

Figure 2. Position of Phe311PseFDH (Phe285CboFDH) (marked by pink color) in ternary complex (PseFDH-NAD+ -azide) (structure 2NAD).