Characterization of microsomal NADPH-dependent aldehyde reductase from rat brain

An NADPH-dependent aldehyde reductase was purified from rat brain microsomes to electrophoretic homogeneity. The purified enzyme had a molecular weight of 75,000 and reduced long chain fatty aldehydes such as octanal and hexadecanal with higher affinity (Km values of 0.21 mM and 0.03 mM, respectively) than for various artificial carbonyl compounds such as p-nitrobenzaldehyde and p-nitroacetophenone (Km values of 0.31 mM and 1.4 mM, respectively). The purified microsomal aldehyde reductase also showed NADPH-cytochrome c reductase activity, and it could not be distinguished from NADPH-cytochrome c reductase in molecular weight (75,000), chromatographic behavior, electrophoretic mobility, or immunological properties. The solubilized microsomal fraction treated with steapsin lost the reductase activity for hexadecanal but not that for cytochrome c. These results suggest that the aldehyde reductase in brain microsomes is identical to NADPH-cytochrome c reductase and that a hydrophobic portion of the NADPH-cytochrome c reductase is required for the reduction of hexadecanal.     

Purification and properties of NADPH-dependent aldehyde reductase from human liver.

An aldehyde reductase (EC 1.1.1.2) from human liver has been purified to homogeneity. The enzyme is NADPH-dependent, prefers aromatic to aliphatic aldehydes as substrates, and is inhibited by barbiturates and hydantoins. The following physicochemical parameters were determined: molecular weight, 36,200; sedimentation coefficient, 2.9 S; Stokes radius, 2.65 nm; isoelectric point, pH 5.3; extinction coefficient at 280 nm, 54,300 M-1 cm-1. Results from polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, gel filtration, and ultracentrifugation suggest a monomeric structure. On molecule of NADPH binds to the enzyme causing a red shift of the coenzyme absorption maximum from 340 to 352 nm. The amino acid composition has been determined and a partial specific volume of 0.74 was computed from these data. An alpha-helicity of 7 and 18% was estimated from the ellipticities at 208 and 222 nm, respectively. Combination of the most reactive thiol group with p-mercuribenzoate does not cause loss of catalytic activity. Inactivation occurs when more than one thiol group is modified. The presence of NADPH or NADP+ prevents loss of activity by thiol modification. The comparison of structural features of aldehyde reductase with other monomeric and oligomeric dehydrogenases suggest similarities of aldehyde reductase with octopine dehydrogenase.     

Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase.

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.     

Disulfiram inhibition of aldehyde reductase from the rat liver

Disulphiram (tetraethylthiuram disulphide teturam, antabus), the known antialcoholic preparation, is studied for its effect on the aldehyde reductase activity (EC 1.1.1.1) in the rats' liver. Apparent Km and V are calculated for acetylaldehyde and NADH as well as Ki of disulphiram relative to the substrate and cofactor of the enzyme. The obtained data permit considering disulphiram a high-specific inhibitor of aldehyde reductase in rats' liver.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.     

Purification and characterization of S-phenacylglutathione reductase from rat liver

An enzyme catalyzing the reduction of S-(2,4-dichlorophenacyl)glutathione to 2',4'-dichloroacetophenone was purified to apparent homogeneity by ion exchange, gel filtration, and hydroxylapatite chromatography from rat hepatic cytosol. The molecular weight was 30,000-37,000. The enzyme is distinct from the glutathione S-transferases, mercaptopyruvate sulfurtransferase, and glyoxalase I. Substrate specificity studies showed that S-phenacylglutathiones are the preferred first substrates and that several thiols (glutathione, mercaptoethanol, L-cysteine, or cysteamine) serve as reducing substrates. The enzyme serves to convert toxic alpha-haloketones, which react rapidly and nonenzymatically with glutathione, to nontoxic alkyl ketones.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

Enzyme localization and orientation of the active site of dissimilatory nitrite reductase from Bacillus firmus

The location of the dissimilatory nitrite reductase and orientation of its reducing site of the Grampositive denitrifier, Bacillus firmus NIAS 237 were examined. Approximately 90% of the total dissimilatory nitrite reductase activity with ascorbate-reduced phenazine methosulfate (PMS) as the electron donor was on the protoplast membrane. Nitrite induced with intact Bacillus cells an alkalinization in the external medium, followed by acidification. The electron transfer inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide, which blocked nitrite reduction with endogenous substrates, inhibited the acidification, but not the alkalinization. Alkalinization was not affected with ascorbate-reduced PMS as the artificial electron donor. This indicated that the alkalinization is not associated with proton consumption outside the cytoplasmic membrane by the extracellular nitrite reduction. The dissimilatory nitrite reductase of B. firmus NIAS 237 was located on the cytoplasmic membrane, and its reducing site is suggested to be on the inner side of this membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - PMS phenazine methosulfate - H⁺/NO _inf2 ^sup- ratio number of consumed protons in the external medium per one ion of NO _inf2 ^sup- reduced     

On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.