NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展

不对称还原胺化反应是制备医药中间体手性胺结构单元的重要反应。目前已有许多不同种类的酶被应用于合成手性胺,其中NAD(P)H依赖型氧化还原酶催化的还原胺化反应最为引人注目,因为其能够一步将潜手性酮化合物完全转化为光学纯的手性胺化合物。文中以亚胺还原酶、氨基酸脱氢酶、冠瘿碱脱氢酶和还原性酮胺化酶为例,从NAD(P)H依赖型氧化还原酶的结构特征、作用机理、分子改造及催化应用等方面,综述了其在不对称还原胺化合成手性胺领域的研究进展。     

Deviations from michaelis-menten behaviour of plant glutamate dehydrogenase with ammonium as variable substrate

A stopped flow kinetic analysis has been performed with a homogeneous protein fraction of plant glutamate dehydrogenase. The enzyme exerts strong negative cooperativity with ammonium as variable substrate. The limiting initial rate constants for low substrate concentrations, as calculated from the kinetic data, indicate that the catalytic efficiency of the enzyme increases at low ammonium concentrations. From this it becomes evident that the reductive amination reaction is highly adaptive to the ammonium environment.     

Steady-state kinetics of glutamate dehydrogenase from Pisum sativum L. mitochondria.

Glutamate dehydrogenase [l-glutamate:NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2]has been purified 487-fold from pea stem mitochondria. The enzyme has a specific activity in the presence of 1 mm CaCl₂ of 54 Enzyme Commission (EC) units. Calcium, manganese, and zinc ions activate the reductive amination reaction. The [Ca²⁺]_0.5 for activation by calcium is 9 μm. The extent of activation by calcium changed during purification and storage. The oxidative deamination was slightly inhibited by calcium. The pH optimum for the reductive amination reaction was 8.0 and for the oxidative deamination was 9.2. At pH 8.0 and in the presence of 1 mm CaCl₂ with the ionic strength held constant the enzyme showed normal kinetics for the reductive amination reaction. Under identical conditions except for the absence of CaCl₂ the oxidative deamination reaction showed normal kinetics for glutamate. There was substrate activation at high NAD⁺ concentrations and these concentrations were avoided in the kinetic analysis. A steady-state kinetic analysis showed that a simple mechanism could not be in effect and a partially random mechanism is proposed.     

Human placental glutamate dehydrogenase - purification - kinetic and regulatory properties - physicochemical studies

Glutamate dehydrogenase (EC. 1.4.1.3) has been purified more than 9,000 times from human placental alcoholic subfractions as a homogenous protein of 55,155 daltons (subunit molecular weight). Kinetic constants for the reverse reaction (reductive amination of α-ketoglutarate) have been shown to be similar to those of the bovine liver enzyme, while the kinetic constants for the forward reaction were markedly different as well as some regulatory properties (lack of activation by ADP in the reverse reaction). The amino acid composition differs from the bovine liver enzyme composition. Furthermore, the tryptic peptide patterns of the placental enzyme and the human liver enzyme have been compared. Besides the low specific activity of this enzyme, the results indicate that human placental glutamate dehydrogenase is closely related to other mammalian glutamate dehydrogenases.     

Short and simple synthesis of (R)- and (S)-4-hydroxypentylaminoacetamide: both enantiomers of the (ω-1)-hydroxylated metabolite of milacemide

Both (R)- and (S)-4-hydroxypentylaminoacetamide have been synthesized by reductive amination of glycinamide on the γ-valerolactols corresponding to (R)- and (S)-γ-valerolactone, respectively. These enantiomeric lactones were readily obtained in high enantiomeric excess (ee) by enzymic porcine pancreatic lipase (PPL) kinetic resolution of rac-methyl γ-hydroxyvalerate. © 1992 Wiley-Liss, Inc.     

Enzymatic synthesis of L-6-hydroxynorleucine

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.     

Characterization of the general anion-binding site in glutamate dehydrogenase-NADPH complex

The reductive amination of alpha-ketoglutarate, catalyzed by bovine liver glutamate dehydrogenase, is inhibited by various anions. Formate and acetate ions are competitive with alpha-ketoglutarate. The pH dependence of the pKi profiles for these anions reveals that they bind to the enzyme-NADPH complex only when an enzymatic residue of pK 8.0 +/- 0.1 in the binary complex is protonated. The ionization of this residue has a delta Hion of 15 +/- 4 kcal/mol. These pK and delta Hion values are not significantly different from those observed in the same complex for the enzyme group which binds the gamma-CO2- of alpha-ketoglutarate and oxalylglycine. It is concluded that formate and acetate also bind to the gamma-carboxylate site in enzyme-NADPH. The Ki values for formate and acetate in a buffer containing 0.1 M phosphate are 20 +/- 4 and 32 +/- 5 mM, respectively, when the pK 8.0 group is fully protonated. Phosphate and trifluoroacetate also show an inhibitory effect, while valerate and sulfate have little effect on the reductive amination rates. The results suggest that specific anions can bind to the gamma-carboxylate site by ionic interactions and alter the kinetic and thermodynamic parameters of the glutamate dehydrogenase-NADPH complex in significant ways.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

NADP-specific isocitrate dehydrogenase in regulation of urea synthesis in rat hepatocytes.

The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.     

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.     

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium.

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and L-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Enzymatic Regeneration of NAD in Enantioselective Oxidation of Secondary Alcohols: Glutamate Dehydrogenase Versus NADH Dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidations, two enzymatic methods for regeneration of the oxidized coenzyme form have been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was catalyzed by xylitol dehydrogenase from the yeast Galactocandida mastotermitis. Reduction of OM₂ (apparently to H₂O) by partially purified NADH dehydrogenase complex from Corynebacterium callunae could drive alcohol oxidations better than reductive amination of EaL-ketoglutarate by glutamate dehydrogenase. A fed-batch procedure was developed that overcame inhibition of glutamate dehydrogenase by α-ketoglutarate (K_is 25 mM), thus increasing the productivity of ketose almost 2-fold. For D-fructose production from D-sorbitol (0.1-0.3M) yields of < 90% and productivities up to 1.30g/(L.h) have been obtained. High conversion of up to 50g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 80-fold higher specificity constant than for D-fructose required complexation of the ketose product with borate. In comparison with reductive amination by glutamate dehydrogenase, advantages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrate (02 versus α-ketoglutarate), and (iii) easier process control (batch versus fed-batch).     

Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.

NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.     

Room-temperature synthesis of L-alanine using the alanine dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Alanine dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-alanine at room temperature was accompanied by no detectable loss of alanine dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.     

Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2)

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an Mr 41,000 in denaturing conditions and an Mr 70,000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized L-valine and L-alpha-aminobutyric acid efficiently, L-isoleucine and L-leucine less efficiently, and did not act on D-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: L-valine, 10.0 mM; NAD+, 0.17 mM; 2-oxoisovalerate, 0.6 mM; and NADH, 0.093 mM. In minimal medium, VDH activity was repressed in the presence of glucose and NH4+, or glycerol and NH4+ or asparagine, and was induced by D- and L-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.     

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L⁻¹ culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis–Menten kinetics, exhibiting apparent mild negative co-operativity with both l -glutamate and NADP⁺, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis–Menten kinetics but both 2-oxoglutarate and NH₄⁺ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.     

Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.     

Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver-Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.     

Partial purification and properties of Halobacterium cutirubrum L-alanine dehydrogenase.

1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.     

Purification and catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis.

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4' of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.