Reduced Nicotinamide Cofactors Research Articles

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.

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.

Characterization of Multicomponent Monosaccharide Solutions Using an Enzyme-Based Sensor Array

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify β-d-glucose and β-d-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.

Preparative regio- and chemoselective functionalization of hydrocarbons catalyzed by cell free preparations of 2-hydroxybiphenyl 3-monooxygenase

Oxygenases are useful catalysts for the selective incorporation of molecular oxygen into hydrocarbons. Here, we report on the application of isolated, cell free 2-hydroxybiphenyl 3-monooxygenase (HbpA) as catalyst for the regio- and chemospecific hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. The catalyst was prepared from recombinant Escherichia coli using expanded bed adsorption chromatography and could be stored without significant loss of activity in lyophilized form. The reaction was performed in an aerated and thermostated simple stirred glass vessel in an aqueous (20% v/v)/organic (80% v/v) reaction medium. This allowed in situ product recovery preventing substrate and product inhibition of the catalyst as well as decay of the labile product 2,3-dihydroxybiphenyl. Enzymatic regeneration of reduced nicotinamide cofactors was achieved using the formate/formate dehydrogenase system. We obtained volumetric productivities of up to 0.45 g l−1 h−1. No significant decrease of productivity was observed within 7 h and more. Product purification (purity 92%) was achieved using solid phase extraction with aluminum oxide followed by crystallization as a polishing step (purity>99%).To our knowledge, these results show for the first time the perspectives of integrated enzyme and cofactor regeneration-based biocatalytic processes in organic/aqueous emulsions, coupled with in situ product recovery.

00/00763 Utilization of hard-coal mining washing wastes and red mud for the production of expanded-clay granulate

Oxygenases are useful catalysts for the selective incorporation of molecular oxygen into hydrocarbons. Here, we report on the application of isolated, cell free 2-hydroxybiphenyl 3-monooxygenase (HbpA) as catalyst for the regio- and chemospecific hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. The catalyst was prepared from recombinant Escherichia coli using expanded bed adsorption chromatography and could be stored without significant loss of activity in lyophilized form. The reaction was performed in an aerated and thermostated simple stirred glass vessel in an aqueous (20% v/v)/organic (80% v/v) reaction medium. This allowed in situ product recovery preventing substrate and product inhibition of the catalyst as well as decay of the labile product 2,3-dihydroxybiphenyl. Enzymatic regeneration of reduced nicotinamide cofactors was achieved using the formate/formate dehydrogenase system. We obtained volumetric productivities of up to 0.45 g l−1 h−1. No significant decrease of productivity was observed within 7 h and more. Product purification (purity 92%) was achieved using solid phase extraction with aluminum oxide followed by crystallization as a polishing step (purity>99%).To our knowledge, these results show for the first time the perspectives of integrated enzyme and cofactor regeneration-based biocatalytic processes in organic/aqueous emulsions, coupled with in situ product recovery.

Cyclohexanone Monooxygenase: A Useful Reagent for Asymmetric Baeyer-Villiger Reactions

Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase catalyzes the Baeyer-Villiger oxidations of a wide variety of ketones to the corresponding lactones, many of which are valuable chiral building blocks. This enzyme accepts cyclobutanones, cyclopentanones, cyclohexanones and several bicyclic ketones as substrates. In most cases, the lactones are produced in high yields and optical purities. This review summarizes all of the published Baeyer-Villiger oxidations catalyzed by cyclohexanone monooxygenase published before mid-1997. An important recent finding is that the enzyme is very proficient at kinetically resolving racemic 2- and 3-substituted cyclohexanones. This provides one of the simplest routes to optically pure substituted £­ caprolactones. Unfortunately, the need to handle the pathogenic Acinetobacter strain as well as the time and expense necessary to purify the enzyme and regenerate the reduced nicotinamide cofactor have hindered the acceptance of cyclohexanone monooxygenase as a synthetic reagent. To overcome these problems, the enzyme has been expressed in baker's yeast so that whole yeast cells can be used for Baeyer-Villiger oxidations in place of the purified enzyme or the Acinetobacter cells. The engineered yeast approach requires no biochemical expertise or equipment beyond that found in a normal laboratory. Finally, active site models of the enzyme are discussed. These allow one to predict the outcome of proposed reactions based on the results summarized here.

Remarkably selective metallized-carbon amperometric biosensors

The use of metallized carbon transducers results in remarkably selective amperometric biosensors. Such focus on the transducer eliminates major electroactive interferences in the first place, and hence circumvents the need for anti-interference layers. The remarkable selectivity of metal-dispersed carbons is attributed to their strong, preferential, electrocatalytic action towards the detection of the enzymatically-liberated hydrogen peroxide and reduced nicotinamide cofactor (NADH). Such activity allows tuning of the operating potential to the region (+ 0.1 to −0.2 V) where unwanted reactions do not occur. This article reviews the development of metallized-carbon biosensors, with particular emphasis on metallized carbon-paste enzyme electrodes, miniaturized devices based on electrochemical deposition of the metal/enzyme layer and metal-dispersed sensor strips.

Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis.

Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24. This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine [Li, L., Falzone, C. J., Wright, P. E., & Benkovic, S. J. (1992) Biochemistry 31, 7826-7833]. An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N epsilon H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks. Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover.

Proof that the biosynthesis of vitamin B12 involves a reduction step in an anaerobic as well as an aerobic organism

Labelling experiments with 2H and 3H prove that as cobyrinic acid is biosynthesised by the anaerobic bacterium Propionibacterium shermanii, its H–19 is derived from HR(and not HS) at C-4 of a reduced nicotinamide cofactor, so demonstrating that the biosynthetic pathway involves a reduction step.

Hepatic metabolism of short-chain bile acids. Inversion of the 3-hydroxyl group of isoetianic acid (3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid) by the adult rat.

The stereospecificity of mechanisms for hepatic transport of short-chain bile acids has been examined by following the hepatic metabolism and biliary secretion of 3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid (isoetianic acid) administered in two different labeled forms to rats prepared with an external biliary fistula. While 93% of the administered [2,2,4,4-3H]isoetianic acid was recovered in bile after 20 h, only 18% of a similar dose of [3 alpha-3H]isoetianic acid was secreted in bile over the same time period. The recovered radioactivity of the latter compound was mainly associated with bile water. With the [2,2,4,4-3H]isoetianic acid, the bulk of the biliary isotope was determined to be in the form of two glucuronide conjugates. Spectral analysis identified these metabolites as the hydroxyl-linked (major) and carboxyl-linked (minor) beta-glucuronides, not of the 3 beta-hydroxy compound administered, but of 3 alpha-hydroxy-5 beta-androstane-17 beta-carboxylic acid (etianic acid), i.e., the products of hydroxyl group inversion. It is concluded that isoetianic acid is efficiently cleared from plasma and conjugated with glucuronic acid after its epimerization to etianic acid. The prevalent, but not complete, loss of the 3-tritium atom and the retention of the 2- and 4-tritium atoms probably indicates a 3-oxo-5 beta-androstane-17 beta-carboxylic acid intermediate with partial return of the label via a limited labeled pool of reduced nicotinamide cofactor.

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.