An NAD(P)(+)-dependent secondary-alcohol dehydrogenase of Alcaligenes eutrophus: Purification, characterization and its application for the production of chiral alcohols

A soil micro-organism identified as Alcaligenes eutrophus capable of utilizing nerolidol, a sesquiterpene alcohol as the sole source of carbon, contains an inducible NAD(P)(+)-linked secondary-alcohol dehydrogenase (SADH), The enzyme was purified 252-fold from crude cell-free extract by a combination of salt precipitation, ion-exchange and affinity-matrix chromatography, Native and SDS/PAGE PAGE of the purified enzyme showed a single protein band and the enzyme appears to be a homotetramer having an apparent molecular mass of 139 kDa comprising four identical subunits of 38.5 kDa, The isoelectric point (pi) of SADH was determined to be 6.2, Depending on pH of the reaction media, the enzyme carried out both oxidation and reductions of various terpenoids and steroids, At pH 5.5, the enzyme catalysed the stereospecific reduction of prochiral ketones to optically active (S)-alcohols and the oxidation reaction was predominated over the former at pH 9.5, NADP(+) and NADPH were respectively preferred over NAD(+) and NADH for oxidation and reduction reactions, The K-m values for testosterone, NADP(+) and NAD(+) were 11.8, 55.6, and 122 mu M respectively, Neither enzyme was significantly inhibited by metal-binding agents, but some thiol-blocking compounds inhibited it, SADH tolerates moderate concentrations of water-miscible organic solvents such as ethanol, methanol, acetone and dioxan, Some of the properties of this enzyme were found to be significantly different from those thus far described.

Interaction Signatures Stabilizing the NAD(P)-Binding Rossmann Fold: A Structure Network Approach

The fidelity of the folding pathways being encoded in the amino acid sequence is met with challenge in instances where proteins with no sequence homology, performing different functions and no apparent evolutionary linkage, adopt a similar fold. The problem stated otherwise is that a limited fold space is available to a repertoire of diverse sequences. The key question is what factors lead to the formation of a fold from diverse sequences. Here, with the NAD(P)-binding Rossmann fold domains as a case study and using the concepts of network theory, we have unveiled the consensus structural features that drive the formation of this fold. We have proposed a graph theoretic formalism to capture the structural details in terms of the conserved atomic interactions in global milieu, and hence extract the essential topological features from diverse sequences. A unified mathematical representation of the different structures together with a judicious concoction of several network parameters enabled us to probe into the structural features driving the adoption of the NAD(P)-binding Rossmann fold. The atomic interactions at key positions seem to be better conserved in proteins, as compared to the residues participating in these interactions. We propose a ``spatial motif'' and several ``fold specific hot spots'' that form the signature structural blueprints of the NAD(P)-binding Rossmann fold domain. Excellent agreement of our data with previous experimental and theoretical studies validates the robustness and validity of the approach. Additionally, comparison of our results with statistical coupling analysis (SCA) provides further support. The methodology proposed here is general and can be applied to similar problems of interest.

Conserved water-mediated recognition and dynamics of NAD(+) (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1 `) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH(2)group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

Requirement Of A Diperoxovanadate-Derived Intermediate for the Interdependent Oxidation Of Vanadyl And Nadh

Oxygen release accompanying oxidation of vanadyl by diperoxovanadate was suppressed on addition of NADH. The added NADH was rapidly oxidized, oxygen in the medium was consumed, and the reaction terminated on exhaustion of either NADH or vanadyl. The consumption of oxygen and disappearance of NADH needed small concentrations of diperoxovanadate to initiate and increased with increase in the concentration of vanadyl and NADH or decrease of pH. The products of the reaction were found to be NAD(+) from NADH and vanadate oligomers from vanadyl and oxygen. The reaction was insensitive to catalase and was not dependent on H2O2. The reaction was inhibited by superoxide dismutase, cytochrome c, EDTA, Mn2+, histidine, and DMPO, but not by hydroxyl radical scavengers such as ethanol and benzoate, The ESR spectrum of the reaction mixture showed the presence of the 1:2:2:1 quartet signal typical of a DMPO-OH adduct, but this was not modified by ethanol, This oxygen radical species, possibly of (OV)-O-. type derived from diperoxovanadate, is proposed to have a role in the reactions of oxygen release and NADH oxidation

Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.

Linalyl Acetate Is Metabolized by Pseudomonas incognita with the Acetoxy Group Intact

Metabolism of linalyl acetate by Pseudomonas incognita isolated by enrichment culture on the acyclic monoterpene alcohol linalool was studied. Biodegradation of linalyl acetate by this strain resulted in the formation of linalool, linalool- 8-carboxylic acid, oleuropeic acid, and A5-4-acetoxy-4-methyl hexenoic acid. Cells adapted to linalyl acetate metabolized linalyl acetate-8-aldehyde to linalool- 8-carboxylic acid, linalyl acetate-8-carboxylic acid, A5-4-acetoxy-4-methyl hexenoic acid, and geraniol-8-carboxylic acid. Resting cell suspensions previously grown with linalyl acetate oxidized linalyl acetate-8-aldehyde to linalyl acetate-8- carboxylic acid, A5-4-acetoxy-4-methyl hexenoic acid, and pyruvic acid. The crude cell-free extract (10,000 g of supernatant), obtained from the sonicate of linalyl acetate-grown cells, was shown to contain enzyme systems responsible for the formation of linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid from linalyl acetate. The same supernatant contained NAD-linked alcohol and aldehyde dehydrogenases involved in the formation of linalyl acetate-8-aldehyde and linalyl acetate-8-carboxylic acid, respectively. On the basis of various metabolites isolated from the culture medium, resting cell experiments, growth and manometric studies carried out with the isolated metabolites as well as related synthetic analogs, and the preliminary enzymatic studies performed with the cellfree extract, a probable pathway for the microbial degradation of linalyl acetate with the acetoxy group intact is suggested.

A hexokinase effect in the inhibition of oxidative phosphorylation in heart muscle mitochondria by Adriamycin

The inhibitory action of the anticancer antibiotic, Adriamycin, on succinate-dependent oxidative phosphorylation in heart mitochondria was markedly potentiated by the presence of hexokinase in the reaction medium. This 'hexokinase effect' was not observed in the oxidation of NAD+-linked substrates, or when liver or kidney mitochondria were used in place of heart mitochondria. These results offer a biochemical explanation for the extreme cardiac toxicity of the drug.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

Metabolism of alpha-terpineol by Pseudomonas incognita

Details of the metabolism of alpha-terpineol by Pseudomonas incognita are presented. Degradation of alpha-terpineol by this organism resulted in the formation of a number of acidic and neutral metabolites. Among the acidic metabolites, beta-isopropyl pimelic acid, 1-hydroxy-4-isopropenyl-cyclohexane-1-carboxylic acid, 8-hydroxycumic acid, oleuropeic acid, cumic acid, and p-isopropenyl benzoic acid have been identified. Neutral metabolites identified were limonene, p-cymene-8-ol, 2-hydroxycineole, and uroterpenol. Cell-free extracts prepared from alpha-terpineol adapted cells were shown to convert alpha-terpineol, p-cymene-8-ol, and limonene to oleuropeic acid, 8-hydroxycumic acid, and perillic acid, respectively, in the presence of NADH. The same cell-free extract contained NAD+ -specific dehydrogenase(s) which converted oleuropyl alcohol, p-cymene-7,8-diol, and perillyl alcohol to their corresponding 7-carboxy acids. On the basis of various metabolites isolated from the culture medium, together with the supporting evidence obtained from enzymatic and growth studies, it appears that P. incognita degrades alpha-terpineol by at least three different routes. While one of the pathways seems to operate via oleuropeic acid, a second may be initiated through the aromatization of alpha-terpineol. The third pathway may involve the formation of limonene from alpha-terpineol and its further metabolism.

Inhibition of mitochondrial oxidative phosphorylation by 2-methyl-4-dimethylaminoazobenzene

The azodye 2-methyl-4-dimethylaminoazobenzene inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. Phosphorylation was more sensitive to the inhibitory action of the azodye than was the oxidation of succinate or ascorbate. The oxidation of NAD+-linked substrate was severely inhibited by the compound. In submitochondrial particles, only NADH oxidation was sensitive. The site of inhibition has been identified to lie between the dehydrogenase flavoprotein and ubiquinone.

Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene

Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H2O2. The rate of H2O2 formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK m value for NADH was found to be 3×10–6 M. The H2O2-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H2O2 generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H2O2. In view of the growing evidence that H2O2 can be involved in metabolic control, the formation of H2O2 by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5prime-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.

Studies on an enzyme reacting with isoniazid fromMycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis H37Rv possesses an enzyme (referred to as ‘Y enzyme’) which catalyses in the presence of INH and NAD, the formation of a product, which turns yellow on acidification. The requirements for the reaction, such as enzyme concentration, INH concentration, etc., have been standardized. The substrate specificity of the enzyme with respect to INH and NAD has been determined. The reaction is specific for the INH-sensitive strain and is totally absent in INH-resistant strains. Furthermore, the ‘Y enzyme’ shows some characteristic features of a peroxidase in its requirement for oxygen and sensitivity to inhibition by various reagents. The requirements of this enzyme which is involved in the action of isoniazid inM. tuberculosis H37Rv is described for the first time.