Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis

Industrial enzymatic reactions requiring 1,4-NAD(P)H₂ to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H₂ from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H₂-dependent enzyme reactions of interest to the industrial biocatalysis community.     

Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis

CDP-D-glucose 4,6-dehydratase catalyzes the conversion of CDP-D-glucose to CDP-4-keto-6-deoxyglucose in an NAD(+)-dependent manner. The product of this conversion is a building block for a variety of primary antigenic determinants in bacteria, possibly implicated directly in reactive arthritis. Here, we describe the solution of the high-resolution crystal structure of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis in the resting state. This structure represents the first CDP nucleotide utilizing dehydratase of the short-chain dehydrogenase/reductase (SDR) family to be determined, as well as the first tetrameric structure of the subfamily of SDR enzymes in which NAD(+) undergoes a full reaction cycle. On the basis of a comparison of this structure with structures of homologous enzymes, a chemical mechanism is proposed in which Tyr157 acts as the catalytic base, initiating hydride transfer by abstraction of the proton from the sugar 4'-hydroxyl. Concomitant with the removal of the proton from the 4'-hydroxyl oxygen, the sugar 4'-hydride is transferred to the B face of the NAD(+) cofactor, forming the reduced cofactor and a CDP-4-keto-d-glucose intermediate. A conserved Lys161 most likely acts to position the NAD(+) cofactor so that hydride transfer is favorable and/or to reduce the pK(a) of Tyr157. Following substrate oxidation, we propose that Lys134, acting as a base, would abstract the 5'-hydrogen of CDP-4-keto-D-glucose, priming the intermediate for the spontaneous loss of water. Finally, the resulting Delta(5,6)-glucoseen intermediate would be reduced suprafacially by the cofactor, and reprotonation at C-5' is likely mediated by Lys134.     

Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design

Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.     

Mechanism and applications of phosphite dehydrogenase

Phosphite dehydrogenase catalyzes the NAD+-dependent oxidation of hydrogen phosphonate (common name phosphite) to phosphate in what amounts to a formal phosphoryl transfer reaction from hydride to hydroxide. This review places the enzyme in the context of phosphorus redox metabolism in nature and discusses the results of mechanistic investigations into its reaction mechanism. The potential of the enzyme as a NAD(P)H cofactor regeneration system is discussed as well as efforts to engineer the cofactor specificity of the protein.     

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.     

Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes

The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.     

Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma

Nicotinamide cofactor-dependent oxidoreductases have been widely employed during the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these biotransformation reactions. Herein, we report the synthesis of an important pharmaceutical intermediate 4-hydroxy-2-butanone (4H2B) via an immobilized in situ cofactor regeneration system composed of NAD(+)-dependent glycerol dehydrogenase (GlyDH) and NAD(+)-regenerating NADH oxidase (nox). Both enzymes were immobilized on functionalized single-walled carbon nanotubes (SWCNTs) through the specific interaction between the His-tagged enzymes and the modified SWCNTs. GlyDH demonstrated ca. 100% native enzyme activity after immobilization. The GlyDH/nox ratio, pH, and amount of nicotinamide cofactor were examined to establish the optimum reaction conditions for 4H2B production. The nanoparticle-supported cofactor regeneration system become more stable and the yield of 4H2B turned out to be almost twice (37%) that of the free enzyme system after a 12-h reaction. Thus, we believe that this non-covalent specific immobilization procedure can be applied to cofactor regeneration system for bioconversions.     

Effect of molecular mobility on coupled enzymatic reactions involving cofactor regeneration using nanoparticle-attached enzymes

Cofactor-dependent multi-step enzymatic reactions generally require dynamic interactions among cofactor, enzyme and substrate molecules. Maintaining such molecular interactions can be quite challenging especially when the catalysts are tethered to solid state supports for heterogeneous catalysis for either biosynthesis or biosensing. The current work examines the effects of the pattern of immobilization, which presumably impacts molecular interactions on the surface of solid supports, on the reaction kinetics of a multienzymic system including glutamate dehydrogenase, glucose dehydrogenase and cofactor NAD(H). Interestingly, particle collision due to Brownian motion of nanoparticles successfully enabled the coupled reactions involving a regeneration cycle of NAD(H) even when the enzymes and cofactor were immobilized separately onto superparamagnetic nanoparticles (124 nm). The impact of particle motion and collision was evident in that the overall reaction rate was increased by over 100% by applying a moderate alternating magnetic field (500 Hz, 17 Gs), or using additional spacers, both of which could improve the mobility of the immobilized catalysts. We further observed that integrated immobilization, which allowed the cofactor to be placed in the molecular vicinity of enzymes on the same nanoparticles, could enhance the reaction rate by 1.8 fold. These results demonstrated the feasibility in manipulating molecular interactions among immobilized catalyst components by using nanoscale fabrication for efficient multienzymic biosynthesis.     

The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase

Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.     

Ontogenesis of oxidative reaction of 17beta-hydroxysteroid dehydrogenase and 11beta-hydroxysteroid dehydrogenase in rat Leydig cells,a histochemical study

The enzyme 17beta-hydroxysteroid dehydrogenase is required for the synthesis and 11beta-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1-90. Using NAD or NADP as the cofactor, 17beta-hydroxysteroid dehydrogenase (substrate: 5-androstene-3beta,17beta-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17beta-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11beta-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17beta-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11beta-hydroxysteroid dehydrogenase in addition to types 1 and 2.     

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.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.     

Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.

Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.     

Ontogenesis of Oxidative Reaction of 17β-hydroxysteroid Dehydrogenase and 11β-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study

The enzyme 17β-hydroxysteroid dehydrogenase is required for the synthesis and 11β-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17β-hydroxysteroid dehydrogenase (substrate: 5-androstene-3β, 17β-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17β-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11β-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17β-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11β -hydroxysteroid dehydrogenase in addition to types 1 and 2.     

Regeneration of the nicotinamide cofactor using a mediator-free electrochemical method with a tin oxide electrode

Tin (IV) oxide was made using an anodization and annealing method and was used as a working electrode in an electrochemical cofactor regeneration reaction. This material was formed with a large surface area, and by changing the preparation conditions, it was possible to control the morphology. Tin oxide has redox properties similar to those of frequently used mediators required for electron transfer between cofactors and an electrode. Therefore, by using tin oxide as a novel electrode, mediator-free electrochemical cofactor regeneration may be possible. Oxidation and reduction of the nicotinamide cofactors, NAD(P)H and NAD(P)⁺, were carried out under various reaction conditions. The results showed a high efficiency for oxidizing NADH over a broad range of pH and temperatures. The oxidation tendency of NADPH was also observed, and it demonstrated a similar reaction tendency as NADH. When using a tin oxide electrode, NAD⁺ was readily reduced to NADH, though the efficiency of this reaction was lower than for NADH oxidation. Oxidation of 2-propanol to acetone was used as a model system using alcohol dehydrogenase and the cofactor regeneration system suggested in this study. The electroenzymatic reaction showed efficient regeneration of NADP⁺ without a mediator.     

Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.     

Ovothiol: a novel thiohistidine compound from sea urchin eggs that confers NAD(P)H-O2 oxidoreductase activity on ovoperoxidase

Sea urchin eggs contain a small molecular weight heat-stable factor that confers cyanide-resistant NAD(P)H-O2 oxidoreductase activity on ovoperoxidase (Turner, E., Somers, C. E., and Shapiro, B. M. (1985) J. Biol. Chem. 260, 13163-13171), the enzyme responsible for cross-linking the extracellular protein coat (fertilization membrane) of the egg. Here we report the isolation of the active cofactor and its identification by ultraviolet, NMR, and mass spectroscopy as a new sulfur-containing amino acid derivative, 1-methyl-alpha N,alpha N-dimethyl-4-thiohistidine, or ovothiol. Ovothiol reacts slowly with atmospheric oxygen or rapidly with micromolar concentrations of H2O2 to form ovothiol disulfide, which is inactive as a cofactor for the ovoperoxidase NAD(P)H oxidase. Reduced active ovothiol is regenerated by treatment with disulfide reductants and shows significant differences in its ultraviolet and NMR spectra from oxidized ovothiol. The oxidoreductase activity of the ovoperoxidase/ovothiol system is similar to that previously characterized with crude cofactor preparations; it is greatly enhanced by Mn2+ and is relatively insensitive to CN-, compared to the peroxidase activity of ovoperoxidase. The ovothiol content of eggs is estimated as 1.8 pmol/egg or an intracellular concentration of 6.8 mM. This concentration exceeds the amount of reductant needed for the CN-(-)insensitive oxygen consumption following fertilization and used in the production of H2O2 for fertilization membrane cross-linking. Whether ovothiol is involved in the cross-linking reaction, protects the egg from damage from H2O2, or has another role in development remains unclear.     

Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol

The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A “bridge” between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals.     

3Alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni: kinetic properties with NAD and its thionicotinamide analogue.

The kinetics of 3alpha-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3alpha-hydroxysteroids from both the C19, C21, and C24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11beta-hydroxy or an 11-oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3alpha-hydroxy group. No such effect could be seen when the 11-hydroxy group was in the alpha-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-depenedent monomeric-dimeric transition of the 3alpha-hydroxysteroid dehydrogenase.