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.     

Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD⁺-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L⁻¹ d⁻¹, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.     

Microwave-enhanced reductive amination via Schiff's base formation for block copolymer synthesis

Reductive amination via Schiff's base formation is a widely used reaction for laboratory and industrial applications ranging from protein immobilization to nanoparticle synthesis. One major limitation of this reaction is the slow kinetics and hence, it can take several days for the reaction to reach completion. Here we demonstrate that electromagnetic microwave can be used to accelerate the rate of reduction amination. To demonstrate proof of concept, we utilized the reductive amination between reducing end of dextran and primary amine from N-Boc-ethylenediamine as a model reaction. The reaction was conducted at room temperature to demonstrate that the enhancement was mainly due to electromagnetic effects of the microwave rather than thermal effects. We show that reductive amination reaction time was reduced from 72 h to 4 h using microwave irradiation. These results indicate non-thermal microwave effects to expedite reductive amination for synthesizing copolymers. The efficient conjugation of dextran using reductive amination provides an important tool for developing biocompatible copolymers using carbohydrates.     

l-Phenylalanine dehydrogenase from Brevibacterium sp. for production of l-phenylalanine by reductive amination of phenylpyruvate

Summary For production of l-phenylalanine the reductive amination of phenylpyruvate, catalyzed by phenylalanine-dehydrogenase was examined. To reach high levels and a sufficient stability of the inducible intracellular enzyme, growth conditions of Brevibacterium sp. are optimized. For continuous production of l-phenylalanine in an enzyme membrane reactor, the kinetic parameters of the partially purified enzyme are determined.In continuous production a space time yield of 37.4 g l-Phe l^-1 d^-1 can be reached.By means of the measured kinetic parameters and simultaneous calculation of the mass balances of all reaction components the behaviour of the reactor can be simulated. For certain conditions the multi-enzyme-system shows multiple steady-states.Abbreviations l-phe l-phenylalanine - phepy phenylpyruvate - PEG polyethylenglycol - pheDH l-phenylalanine dehydrogenase     

Efficient regeneration of NADPH using an engineered phosphite dehydrogenase

The in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals. Here, we report the engineering of a highly stable and active mutant phosphite dehydrogenase (12x-A176R PTDH) from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regeneration system in an enzyme membrane reactor. Two practically important enzymatic reactions including xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis were used as model systems, and the mutant PTDH was directly compared to the commercially available NADP(+)-specific Pseudomonas sp. 101 formate dehydrogenase (mut Pse-FDH) that is widely used for NADPH regeneration. In both model reactions, the two regeneration enzymes showed similar rates of enzyme activity loss; however, the mutant PTDH showed higher substrate conversion and higher total turnover numbers for NADP(+) than mut Pse-FDH. The space-time yields of the product with the mutant PTDH were also up to fourfold higher than those with mut Pse-FDH. In particular, a space-time yield of 230 g L(-1) d(-1) xylitol was obtained with the mutant PTDH using a charged nanofiltration membrane, representing the highest productivity compared to other existing biological processes for xylitol synthesis based on yeast D-xylose converting strains or similar in vitro enzyme membrane reactor systems.     

Inhibition by N'-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in alpha-ketoglutarate amination

The effect of N'-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the alpha-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K(i), estimated as 199 micromol l(-1) at 25 degrees C and pH 8.0.     

Influence of glutamate dehydrogenase activity on L-proline synthesis

The influence of glutamate dehydrogenase activity on the production of L-proline in the producer strain Brevibacterium flavum AP111 was studied. Transformants of the Brevibacterium flavum strain AP111 were obtained with the use of a recombinant plasmid previously constructed by us, pVOG10. It includes the encoding sequence of the gdh gene, which is responsible for the synthesis of glutamate dehydrogenase. The production of L-proline in transformants was shown to have increased by more than 40%. The results can serve as the basis for the use of this approach to improve strains producing other amino acids of the glutamine family.     

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。     

The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

Improved procedure for direct coupling of carbohydrates to proteins via reductive amination

Carbohydrate-protein conjugates are utilized extensively in basic research and as immunogens in a variety of bacterial vaccines and cancer vaccines. As a result, there have been significant efforts to develop simple and reliable methods for the construction of these conjugates. While direct coupling via reductive amination is an appealing approach, the reaction is typically very inefficient. In this paper, we report improved reaction conditions providing an approximately 500% increase in yield. In addition to optimizing a series of standard reaction parameters, we found that addition of 500 mM sodium sulfate improves the coupling efficiency. To illustrate the utility of these conditions, a series of high mannose BSA conjugates were produced and incorporated into a carbohydrate microarray. Ligand binding to ConA could be observed and apparent affinity constants ( K ds) measured using the array were in good agreement with values reported by surface plasmon resonance. The results show that the conditions are suitable for microgram-scale reactions, are compatible with complex carbohydrates, and produce biologically active conjugates.     

Synthesis of lactoside glycodendrons using photoaddition and reductive amination methodologies

Carbohydrate-based divalent and tetravalent lactoside glycodendrons were constructed in a convergent manner. The dendrons were synthesized beginning with the photoaddition of hepta-O-acetyl-1-thio-beta-lactose, in an anti-Markovnikov manner, to a bis-allyl AB2 trisaccharide to form a divalent dendron. Following two nearly quantitative deprotection steps, the divalent lactoside was coupled to another AB2 trisaccharide by reductive amination to afford a tetravalent dendron. These paucivalent compounds were characterized by NMR spectroscopy and mass spectrometry.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms

1. Aspergillus nidulans, Neurospora crassa and Escherichia coli were grown on media containing a range of concentrations of nitrate, or ammonia, or urea, or l-glutamate, or l-glutamine as the sole source of nitrogen and the glutamate dehydrogenate and glutamine synthetase of the cells measured. 2. Aspergillus, Neurospora and Escherichia coli cells, grown on l-glutamate or on high concentrations of ammonia or on high concentrations of urea, possessed low glutamate dehydrogenase activity compared with cells grown on other nitrogen sources. 3. Aspergillus, Neurospora and Escherichia coli cells grown on l-glutamate possessed high glutamine synthetase activity compared with cells grown on other nitrogen sources. 4. The hypothesis is proposed that in Aspergillus, Neurospora and Escherichia colil-glutamate represses the synthesis of glutamate dehydrogenase and l-glutamine represses the synthesis of glutamine synthetase. 5. A comparison of the glutamine-synthesizing activity and the gamma-glutamyltransferase activity of glutamine synthetase in Aspergillus and Neurospora gave no indication that these fungi produce different forms of glutamine synthetase when grown on ammonia or l-glutamate as nitrogen sources.     

Recovery of free oligosaccharides from derivatives labeled by reductive amination

This study examined chemical regeneration of free oligosaccharides from their fluorescent derivatives prepared by reductive amination with various aromatic amines. Maltose derivatives of ethyl 4-aminobenzoate (p-ABEE), 2-aminobenzonitrile (o-ABN), 4-aminobenzonitrile (p-ABN), 7-amino-4-methylcoumarin (AMC), 2-aminobenzoic acid (o-ABA), 2-aminobenzamide (o-ABAD), 2-aminopyridine (AP), and 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) were incubated at 30 degrees C with an aqueous solution of hydrogen peroxide/acetic acid. Recoveries of maltose from p-ABEE, p-ABN, and AMC derivatives were fairly good and gave approximately 90% of maltose. Recoveries of maltose from its o-substituted aniline (o-ABA, o-ABAD, and o-ABN) derivatives were 5-40%, but maltose was unrecoverable from AP and ANTS derivatives. Nevertheless, prior treatment of an AP derivative with cyanogen bromide enabled the regeneration of maltose in high yields. As an application, p-ABEE-labeled N-glycans from some glycoproteins separated on an amide column were identified by converting peak components to their AP derivatives via free saccharides and following mapping by reversed-phase chromatography.