Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides

A protein with a mol. mass of 51,000 (ThcE) that was induced in Rhodococcus sp. N186/21 during assimilation of thiocarbamate herbicides, atrazine, ethanol, propanol, glycerol, propionaldehyde or ethanolamine was identified by two-dimensional electrophoresis. The thcE gene was cloned and sequenced. The deduced amino acid sequence revealed ThcE as a member of group III alcohol dehydrogenases. ThcE displayed strong homology with sequenced subunit fragments of the homodecameric N,N-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductases (MNO) of Amycolatopsis methanolica and Mycobacterium gastri. N-Terminal sequence analysis of purified MNO from Rhodococcus sp. NI86/21 confirmed the identity with ThcE. When overproduced in Escherichia coli, ThcE was insoluble and no MNO activity was detected.Abbreviations BSM Basal salt medium - EPTC S-ethyl dipropylthiocarbamate - MDH methanol dehydrogenase - MNO methanol - NDMA oxidoreductase - NDMA N,N-dimethyl-4-nitrosoaniline     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca²⁺-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La³⁺-dependent. Despite the absence of Ca²⁺ in the medium strain AM1 was able to grow on methanol in the presence of La³⁺. Addition of La³⁺ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La³⁺, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La³⁺ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca²⁺, but was able to grow after supplementation with La³⁺. Taken together, these results show that XoxF1 participates in methanol metabolism as a La³⁺-dependent MDH in strain AM1.     

Anisakis,Phocanema, Contracaecum, and Sulcascaris Spp.: Electrophoresis and thermostability of alcohol and malate dehydrogenases from larvae

Electrophoretic surveys were conducted on individual larvae of four anisakine nematode genera: Anisakis, Phocanema, Contracaecum, and Sulcascaris. The larval worms were obtained from a variety of fish and molluscan hosts from widely dispersed geographic regions. Of several enzymes detected, constant and apparently species-specific electrophoretic patterns were obtained for alcohol dehydrogenase (ADH, alcohol:NAD oxidoreductase, EC 1.1.1.1) and malate dehydrogenase (MDH, l-malate: NAD oxidoreductase, EC 1.1.1.37). ADH, in all but Sulcascaris sp., possessed two isozymes, the slower of which was sensitive to temperature and inhibitors. Failure of preelectrophoretic treatment with NAD to cause interconversion of these isozymes suggests that they are products of separate genetic loci. Both isozymes were maximally active with isopropanol, sec-butanol, and amyl alcohol. Within a given species, ADH showed negligible variation (i.e., apparent genetic polymorphism) with respect to individual larvae, site of larvae in the host, or geographical origin of the host. MDH from Anisakis, Sulcascaris, and Phocanema spp. possessed one, two, and three bands of activity, respectively; MDH is highly thermostable in Anisakis sp. but not in the other species.     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase–ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E·NAD⁺) and three ternary (E·NAD⁺·acetone, E·NAD⁺·3-pentanone and E·NAD⁺·cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Molecular,biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.     

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)⁺ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD⁺ dependent enzyme and an NAD⁺ regenerating enzyme (H₂O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD⁺-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD⁺ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)⁺ for catalysis.     

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act.     

Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k_cat = 823 s⁻¹, K_m = 28.0 mM, and k_cat/K_m = 29.4 mM⁻¹ s⁻¹) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.     

Soluble and chloroplast malate dehydrogenase isoenzymes of Triticum aestivum

Three isoenzymes of malate dehydrogenase have been isolated from 9-day-old wheat shoots. The microbody (peroxisome) and chloroplast MDH are similar in their electrophoretic behaviour. The mitochondrial MDH, soluble MDH and chloroplast MDH differ in K_m values for malate and NAD. The activity of MDH isoenzymes with NAD⁺-analogues as substrate was in the order 3-AP-NAD⁺ > 3-AP-deam NAD⁺ > NAD⁺ > TN-NAD⁺ and deam NAD⁺. The thermal stabilities of the isoenzymes were significantly different: C-MDH > m-MDH > S-MDH.     

Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Purification and characterization of methanol dehydrogenase of Methylobacterium nodulans rhizosphere phytosymbionts

Methanol dehydrogenase (MDH) of the facultative methylotrophic phytosymbiont Methylobacterium nodulans has been purified for the first time to an electrophoretically homogeneous state and characterized. The native protein with a molecular mass of 70 kDa consists of large (60 kDa) and small (6.5 kDa) subunits. The purified protein displayed a spectrum identical to that of pyrroloquinoline quinone (PQQ)-containing MDH, pI 8.7, pH optimum in the range 9–10. The enzyme was inactive in the absence of ammonium or methylamine and exhibited a wide substrate specificity with regard to C₁–C₅ alcohols with the high-est affinity to methanol (K _M = 70 μM), but it did not oxidize benzyl and secondary alcohols. The apparent K _M values to primary alcohols increased with the length of the carbon chain. The enzyme was characterized by a high stability level even in the absence of a substrate. An immobilized enzyme was used for amperometric methanol detection.     

Purification and biochemical properties of genetically defined malate dehydrogenase in maize

Malate dehydrogenase of maize exists in multiple molecular forms (isozymes). In strain W64A, two soluble forms (s-MDH), five mitochondrial forms (m-MDH), and two glyoxysomal forms (g-MDH) were found in etiolated seedlings. The s-MDHs and m-MDHs were prepared in highly purified form. Using these purified isozymes, experiments with reducing agents (100 mm mercaptoethanol), low pH (2.0), and high salt cocn (7.5 m guanidine-HCl), along with genetic data, have eliminated the possibility of conformational alterations as an explanation for MDH multiplicity in maize; the MDH isozymes are genetically determined. Biochemical properties for each of the seven MDH isozymes were examined. Molecular weight, pI, pH optimum, thermolability, and K_m for oxaloacetate, malate, NAD, and NADH at different pH values were determined for each isozyme. Different kinetics of substrate inhibition (oxaloacetate) and coenzyme inhibition (NAD) were observed for the different isozymes. Effects of NAD analogs, chelating agents, reducing agents, metal ions, and TCA cycle acids on the enzymatic activity of these isozymes were tested. Based on the physical and kinetic properties observed, the maize malate dehydrogenase isozymes can be classified into four groups: s-MDH¹; s-MDH²; the two most anodal m-MDHs; and the three most cathodal m-MDHs. Since strain W64A is highly inbred, our data along with our previous and simultaneous genetic analysis suggest that multiple genes are involved in the expression of maize malate dehydrogenase isozymes.     

Prevention of in Vitro thermal aggregation and inactivation of foreign proteins by the hyperthermophilic group II chaperonin α-subunit from Aeropyrum pernix K1

In this study, we report that the recombinant α subunit chaperonin protein (ApCpnA) from Aeropyrum pernix K1 can efficiently prevent the thermal aggregation and inactivation of foreign model proteins, such as citrate synthase (CS) from= porcine heart, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (four 37.5 kDa subunits), and malate dehydrogenase (MDH) from Thermus flavus (two 67 kDa subunits)K=In the presence of ApCpnA and ATP, the thermal aggregation of CS and ADH were prevented by 90 and 65%, respectively, at each 43 and 50°C. Also, the activities of CS, ADH, and MDH under the thermal inactivation conditions were stably maintained at higher than 80% by addition of ApCpnA and ATP, while the activities of those enzymes in the absence of ApCpnA and ATP were dramatically inactivated and decreased below 20% within 30 min. Based on these results, we propose that the α subunit chaperonin from the hyperthermophilic archaeon, A. pernix K1 can be utilized to enhance the durability and cost effectiveness of high-temperature biocatalysts.     

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP⁺-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Improved synthesis of chiral alcohols with <Emphasis Type="Italic">Escherichia coli</Emphasis> cells co-expressing pyridine nucleotide transhydrogenase,NADP<Superscript>+</Superscript>-dependent alcohol dehydrogenase and NAD<Superscript>+</Superscript>-dependent formate dehydrogenase

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD⁺ and NADPH. The pnta and pntb genes encoding for the - and -subunits were cloned and co-expressed with NADP⁺-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.