Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Dichlorophenolindophenol-linked formate dehydrogenase of the methanol-utilizing Mycobacterium gastri MB19

Abstract 2,6-Dichlorophenolindophenol (DCIP)-linked formate dehydrogenase activity has been demonstrated for the first time in the cell-free extract of a methylotrophic mycobacterium, Mycobacterium gastri MB19. The enzyme was produced when the strain was cultivated with methanol, glucose or mannitol as a carbon source, whereas no enzyme production occurred with other multi-carbon compounds. The enzyme was located in the particulate fraction. Although the enzyme was unstable on preservation at 4° C in potassium phosphate buffer (pH 7.0), it was stabilized under acidic conditions (pH 5.0). Glycerol and EDTA were also effective for the enzyme's stability. The optimum pH and temperature for the enzyme's activity were 7.0° and 55° C, respectively.     

Polysulfide reductase and formate dehydrogenase from Wolinella succinogenes contain molybdopterin guanine dinucleotide

Pterin derivatives were extracted from formate dehydrogenase and from polysulfide reductase of Wolinella succinogenes and converted to 6-carboxypterin. The amounts of 6-carboxypterin were consisted with the molybdenum content of the enzymes. The bis(carboxamidomethyl) derivatives of the cofactors showed absorption spectra that were identical with that of the corresponding molybdopterin guanine dinucleotide derivative (cam MGD). After hydrolysis of the derivatives with nucleotide pyrophosphatase in the presence of alkaline phosphatase, guanosine was formed together with a compound showing the properties of dephospho-bis(carboxamidomethyl)-molybdopterin. It is conluded that both formate dehydrogenase and polysulfide reductase of W. succinogenes contain molybdopterin guanine dinucleotide.Abbreviations MPT molybdopterin - MGD molybdopterin guanine dinucleotide - cam MPT bis(carboxyamidomethyl)-molybdopterin - cam MGD bis(carboxyamidomethyl)-molybdopterin guanine dinucleotide     

Escherichia coli formate dehydrogenase mutants with altered selenopolymer profiles

Four classes of Escherichia coli mutants deficient in either or both of their anaerobic selenium-containing formate dehydrogenases (FDH) were isolated. A class I mutant devoid of FDH_H activity specifically linked to benzyl viologen (BV) produced a small amount of the FDH_H 80,000 dalton selenopeptide. Three class II mutants were deficient in FDH_N activity specifically linked to phenazine methosulfate (PMS) and exhibited a selenopeptide doublet rather than the FDH_N 110,000 dalton selenosubunit. Three class III mutants were selenium incorporation deficient and did not exhibit either FDH activity or ⁷⁵Selabeled selenopolymers. A class IV mutant was devoid of PMS-linked FDH_N activity; neither its FDH_N 110,000 dalton selenosubunit nor its BV-linked FDH_H activity was fully regulated by nitrate.Abbreviations FDH formate dehydrogenase - BV benzyl viologen - MV methyl viologen - PMS phenazine methosulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 Å resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.     

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO₂ reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO₂ reduction.     

Effect of surface electrostatic interactions on the stability and folding of formate dehydrogenase from Candida methylica

NAD⁺-dependent formate dehydrogenase (FDH-EC 1.2.1.2) is an important enzyme to regenerate valuable NADH required by NAD⁺-dependent oxidoreductases in enzyme catalysis. The limitation in the thermostability of FDH enzyme is a crucial problem for development of biotechnological and industrial processes, despite of its advantages. In this study, to investigate the contribution of surface electrostatic interaction to the thermostability of FDH from Candida methylica (cmFDH) N187E, H13E, Q105R, N300E, N147R N300E/N147R, N187E/Q105R, N187E/N147R,Y160R, Y302R, Y160E and Y302E mutants were designed using a homology model of cmFDH based on Candida boidinii (cb) by considering electrostatic interactions on the protein surface. The effects of site-specific engineering on the stability of this molecule was analyzed according to minimal model of folding and assembly reaction and deduced equilibrium properties of the native system with respect to its thermal and denaturant sensitivities. It was observed that mutations did not change the unfolding pattern of native cmFDH and increased numbers of electrostatic interactions can cause either stabilizing or destabilizing effect on the thermostability of this protein. The thermodynamic and kinetic results suggested that except relatively improved mutants, three out of the nine single mutations increased the melting temperature of cmFDH enzyme.     

The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor

Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes.     

Asymmetric synthesis of a fluoxetine precursor with an artificial fusion protein of a ketoreductase and a formate dehydrogenase

Herein we describe the kinetic characterization of a fusion protein from the 3-ketoacyl-[acyl-carrier-protein]-reductase (KR) from Synechococcus PCC 7942 and a mutant formate dehydrogenase from Mycobacterium vaccae N10 (MycFDH). Upon purification, a specific proteolytic cleavage of the MycFDH was observed. The cleavage site was elucidated, which is ubiquitously spread among prokaryotic FDHs. After depletion of the cleavage site the correct, full length fusion protein was obtained. In asymmetric reductions of ethylbenzoyl acetate (EBA) this fusion protein performed equal or even better than the free enzymes, yielding up to 39% more of the fluoxetine precursor ethyl-(S)-3-hydroxy-3-phenylpropanoate ((S)-HPPE). The rate acceleration is due to an improved K_m,EBA of the KR subunit.     

Chromogenic assessment of the three molybdo-selenoprotein formate dehydrogenases in Escherichia coli

Escherichia coli synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.     

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.     

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions.     

Transition metal requirement to express high level NAD+-dependent formate dehydrogenase from a serine-type methylotrophic bacterium

Abstract Formate dehydrogenase (EC 1.2.1.2) from an aerobic organism was found to be metal-dependent. This NAD⁺-dependent enxyme required the presence of tungsten or molybdenum to express high enzyme levels in the facultative methylotrophic Methylobacterium sp. RXM. The apparent V _max of the reaction increased 22-fold in a tungstate-containing medium when compared with a non-metal-supplemented growth medium. The absence of those metals in the culture medium resulted in the partial loss of an energy-yielding step and approx. 50% decrease in the cell yield was observed. Moreover, formate accumulated in the extracellular medium and culture pH dropped. Tungsten produced a higher stimulation of formate dehydrogenase activity than that obtained with molybdenum for batch cultivation of Methylobacterium sp. RXM.     

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

The 1.6 A crystal structure of E. coli argininosuccinate synthetase suggests a conformational change during catalysis.

BACKGROUND: Argininosuccinate synthetase (AS) is the rate-limiting enzyme of both the urea and arginine-citrulline cycles. In mammals, deficiency of AS leads to citrullinemia, a debilitating and often fatal autosomal recessive urea cycle disorder, whereas its overexpression for sustained nitric oxide production via the arginine-citrulline cycle leads to the potentially fatal hypotension associated with septic and cytokine-induced circulatory shock. RESULTS: The crystal structure of E. coli AS (EAS) has been determined by the use of selenomethionine incorporation and MAD phasing. The structure has been refined at 1.6 A resolution in the absence of its substrates and at 2.0 A in the presence of aspartate and citrulline (EAS*CIT+ASP). Each monomer of this tetrameric protein has two structural domains: a nucleotide binding domain similar to that of the "N-type" ATP pyrophosphatase class of enzymes, and a novel catalytic/multimerization domain. The EAS*CIT+ASP structure clearly describes the binding of citrulline at the cleft between the two domains and of aspartate to a loop of the nucleotide binding domain, whereas homology modeling with the N-type ATP pyrophosphatases has provided the location of ATP binding. CONCLUSIONS: The first three-dimensional structures of AS are reported. The fold of the nucleotide binding domain confirms AS as the fourth structurally defined member of the N-type ATP pyrophosphatases. The structures identify catalytically important residues and suggest the requirement for a conformational change during the catalytic cycle. Sequence similarity between the bacterial and human enzymes has been used for providing insight into the structural and functional effects of observed clinical mutations.     

Assembly of graphene oxide‐formate dehydrogenase composites by nickel‐coordination with enhanced stability and reusability

Featuring unique planar structure, large surface area and biocompatibility, graphene oxide (GO) has been widely taken as an ideal scaffold for the immobilization of various enzymes. In this regard, nickel‐coordinated graphene oxide composites (GO‐Ni) were prepared as novel supporters for the immobilization of formate dehydrogenase. The catalytic activity, stability and morphology were studied. Compared with GO, the enzyme loading capacity of GO‐Ni was enhanced by 5.2‐fold, besides the immobilized enzyme GO‐Ni‐FDH exhibited better thermostability, storage stability and reuse stability than GO‐FDH. GO‐Ni‐FDH retained 40.9% of its initial activity after 3 h at 60°C, and retained 31.4% of its initial relative activity after 20 days’ storage at 4°C. After eight times usages, GO‐Ni‐FDH maintained 63.8% of its initial activity. Mechanism insights of the multiple interactions of enzyme with the GO‐Ni were studied, considering coordination bonds, hydrogen bonds, electrostatic forces, coordination bonds, and etc. A practical and simple immobilization strategy by metal ions coordination for multimeric dehydrogenase was developed.     

Stereoselective synthesis of opine-type secondary amine car☐ylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes

The substrate specificity of the recently discovered enzyme, opine dehydrogenase (ODH) fromArthrobacter sp. strain 1C for amino donors in the reaction that forms secondary amines using pyruvate as a fixed amino acceptor is examined. The enzyme was active toward short-chain aliphatic (S)-amino acids and those substituted with acyloxy, phosphonooxy, and halogen groups. The enzyme was named N-[1-(R)-(car☐yl)ethyl]-(S)-norvaline: NAD⁺ oxidoreductase (L-norvaline forming). Other substrates for the enzyme were 3-aminobutyric acid and (S)-phenylalaninol. Optically pure opine-type secondary amine car☐ylic acids were synthesized from amino acids and their analogs such as (S)-methionine, (S)-isoleucine, (S)-leucine, (S)-valine, (S)-phenylalanine, (S)-alanine, (S)-threonine, (S)-serine, and (S)-phenylalaninol, and -keto acids such as glyoxylate, pyruvate, and 2-oxobutyrate using the enzyme, with regeneration of NADH by formate dehydrogenase (FDH) fromMoraxella sp. C-1. The absolute configuration of the nascent asymmetric center of the opines was of the (R) stereochemistry with > 99.9% e.e. One-pot synthesis of N-[1-(R)-(car☐yl)ethyl]-(S)-phenylalanine from phenylpyruvate and pyruvate by using ODH, FDH, and phenylalanine dehydrogenase (PheDH) fromBacillus sphaericus, is also described.