Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Physarum polycephalum malate dehydrogenase: inhibitor analyses of the mitochondrial and supernatant isozymes

The effects of naturally occurring metabolites were tested on the malate dehydrogenase (L-malate: NAD+oxidoreductase, EC 1.1.1.37) isozymes from the eucaryotic protist Physarum polycephalum. Several of the Krebs cycle intermediates were inhibitors for each isozyme indicating that a similar catalytic process was involved for both forms. The metabolites ATP, ADP, and AMP were inhibitors competitive with NAD for the mitochondrial isozyme but not the supernatant form. Several other nucleoside phosphates had no effects. Tests of protein sulfhydryl, arginine- and tyrosine-modifying reagents revealed a similar functional sensitivity by both isozymes to these reagents. Those results are compared with data on isozymes from more complex tissue with comments on the physiological significance of those combined data.     

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.     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

Pyruvate dehydrogenase activity during ripening of hamlin oranges

Pyruvate dehydrogenase (PD) was examined as a possible regulatory enzyme in the decline in aerobic respiration and increase in anaerobic metabolism in the ripening Hamlin orange. Oranges were harvested weekly over the growing season from October to February. Juice vesicles were excised and analysed for PD and the cofactors, NADH, NAD, ATP and ADP as well as for reduced and oxidized ubiquinone. PD levels increased slightly from October through February. The cofactor ratio of ATP/ADP increased slightly during that period, NADH/NAD increased more than 2-fold and ubiquinone became more reduced. Data suggest that PD could function as a regulatory enzyme in pyruvate metabolism and that either substrate levels of NAD dehydrogenases increased beyond the capacity of the respiratory pathway to adjust to a higher flux, or the oxidative function of the pathway declined over the season.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Distinction between NAD- and NADH-binding forms of mitochondrial malate dehydrogenase as shown by inhibition with thenoyltrifuoroacetone.

The inhibition of mitochondrial malate dehydrogenase (L-malate : NADH oxidoreductase, EC 1.1.1.37) by 2-thenoyltrifluoroacetone (TTFA) was investigated at pH 8.0 where both forward and backward reactions can be measured. The inhibition with respect to malate is non-competitive at finite NAD concentrations. Increasing the NAD concentrations lowers the slope of the double reciprocal plot so that at infinite NAD the inhibition is uncompetitive. The inhibition with respect to oxaloacetate is non-competitive. Increasing the NADH concentration lowers the slope and intercept of the double reciprocal plot so that at infinite NADH the inhibition is nil. The inhibition with respect to NADH is competitive, whatever the oxaloacetate concentrations are. The inhibition with respect to NAD, at all malate concentrations, is non-competitive. This pattern of inhibition is incompatible with any model assuming that NAD and NADH reacts with identical forms of the enzyme. On the other hand the reciprocating compulsory ordered mechanism, where the two subunits of the dimeric enzyme are working in concert, can account for all the experimental results. It is concluded that NAD and NADH bind to different forms of the enzyme separated by reversible steps. Only one form (see text), the one which binds NADH, can react to form the dead end complex (see text). The similarity between mechanism of inhibition by thenoyltrifluoroacetone and other hydrophobic inhibitors of malate dehydrogenase is discussed.     

High-resolution structures of formate dehydrogenase from Candida boidinii

The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD(+)-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies.     

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis.

The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.     

Human glutamic-gamma-semialdehyde dehydrogenase. Kinetic mechanism.

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.     

Growth of Candida boidinii on methanol and the activity of methanol-degrading enzymes as affected from formaldehyde and methylformate

Formaldehyde and methylformate affect the growth of Candida boidinii on methanol and the activity of methanol-degrading enzymes. The presence of both intermediates in the feeding medium caused an increase in biomass yield and productivity and a decrease in the specific rate of methanol consumption. In the presence of formaldehyde, the activity of formaldehyde dehydrogenase and formate dehydrogenase was essentially increased, whereas the activity of methanol oxidase was decreased. On the contrary, the presence of methylformate caused an increase of the activity of methanol oxidase and a decrease of the activity of formaldehyde dehydrogenase and formate dehydrogenase. Interpretations concerning the yeast behavior in the presence of intermediate oxidation products were considered and discussed.     

A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351.

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.     

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.     

Stabilization of NADH dehydrogenase in mitochondria by adenosine phosphates

It is known that one of the reasons leading to the development of neuroligical disorders, such as Parkinson’s disease, is the damage of the mitochondrial NADH dehydrogenase. We suggest that it happens when NADH dehydrogenase loses connection with its coenzyme flavine mononucleotide (FMN) in the active center. This process is blocked by the enzyme substrate NADH or by the reaction product NAD. In this work we have developed a method based on fluorescence spectroscopy to monitor the stability of FMN in isolated rat liver mitochondria. It was observed that this process is strongly blocked by adenine analogs ATP, ADP, and AMP. Adenine, adenosine, NADPH, nicotine amide, and nicotine acid did not prevent the FMN loss. The obtained data could be used as a basis for construction of synthetic analogues of adenosine phosphates for the treatment of mitochondrial diseases.     

Protein engineering of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.     

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.     

Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

Molecular modeling of alcohol dehydrogenase suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30 degrees C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD+ and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver beta enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD+ complex limiting for turnover with ethanol.