Cofactor recycling in liquid membrane-enzyme systems.

In contrast to other entrapment techniques, hydrocarbon-based liquid surfactant membranes have been shown to effectively retain NADH and NAD⁺. The activities of an immobilized yeast alcohol dehydrogenase (ADH) - NAD⁺ system and of a coupled cofactor recycling system involving ADH, diaphorase and ferricyanide were examined by determining the extent of both ethanol consumption and acetaldehyde accumulation in the external aqueous solution. The results establish suitability of the liquid membrane system for the immobilization of enzyme systems involving $\text{in}$-$\text{situ}$ cofactor regeneration.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

Synthesis of a Highly Substituted N-Linked Immobilized NAD Derivative Using a Rapid Solid-Phase Modular Approach: Suitability for Use with the Kinetic Locking-on Tactic for Bioaffinity Purification of NAD-Dependent Dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD⁺-dependent dehydrogenases. Specifically, the synthesis of highly substituted N⁶-linked immobilized NAD⁺ derivatives is described using a rapid solid-phase modular approach. Other modifications of the N⁶-linked immobilized NAD⁺ derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 Å) in an attempt to minimize nonbiospecific interactions. Analysis of the N⁶-linked NAD⁺ derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S⁶-linked immobilized NAD⁺ derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. -phenylalanine dehydrogenase ( -PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and -phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD⁺-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S⁶-linked immobilized cofactor derivatives than for the new N⁶-linked derivatives. In contrast, the NAD⁺-dependent phenylalanine dehydrogenase showed no affinity for the S⁶-linked immobilized NAD⁺ derivative, but was locked-on strongly to the N⁶-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N⁶-linked immobilized NADP⁺ derivative in the presence of -phenylalanine. This differential locking-on of NAD⁺-dependent dehydrogenases to N⁶-linked and S⁶-linked immobilized NAD⁺ derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD⁺ immobilized via N⁶-linkage as compared to thiol-linkage.     

Metabolisme du γ-aminobutyrate chez Agaricus bisporus III. La succinate-semialdehyde:NAD(P)+ oxydoreductase

Metabolism of γ-Aminobutyrate in Agaricus bisporus. III. The Succinate-Semialdehyde: NAD (P)⁺ Oxidoreductase. The succinate-semialdehyde:NAD(P)⁺ oxidoreductase (E.C. 1.2.1.16) is responsible for the second step in the catabolism of γ-aminobutyrate: the irreversible enzymatic conversion of succinic semialdehyde (SSA) to succinate. Succinate semialdehyde dehydrogenase was extracted from mitochondrial fraction of fruit-bodies of Agaricus bisporus Lge. The mitochondrial pellet was sonicated and centrifuged at 110,000 g; the supernatant obtained was designated the “crude extract”. The enzyme was extremely unstable on storage, unless 1 mM EDTA and 20% glycerol were added. Kinetic studies were carried out at 30°C, and the formation of NADH or NADPH was followed by measuring increase of absorbance at 340 nm with a spectrophotometer. The dehydrogenase was completely inactive when the reaction was run in the absence of thiol and was more active with NAD⁺ than with NADP⁺. In the “crude extract” the activity with NADP⁺ had a pH optimum between 8.6 and 9.1 and the K_m values for SSA and NADP⁺ were 2.0 × 10^?4M and 1.4 × 10^?4M respectively. The pH optimum with NAD⁺ was found between 8.6 and 8.8 and the K_m value for SSA is 4.8 × 10^?4M and for NAD⁺ 2.0 × 10^?3M. With NAD⁺, the kinetic values (pH, K_m) of the “crude extract” chromatographed on hydroxylapatite were unchanged. Inhibition by thiamine pyrophosphate (TPP) was uncompetitive with respect to NAD⁺, those by malate, ATP, ADP and NADPH non-competitive and that by NADH competitive. These results and the fact that activity with NAD⁺ was lost more slowly than with NADP⁺ indicate the possibility of at least two mitochondrial succinate-semialdehyde dehydrogenases, even though the activities of this enzyme assayed with NAD⁺ and NADP⁺ respectively were not able to be separated from each other by hydroxylapatite column chromatography. Some speculations on the metabolic regulation of this dehydrogenase and considerations on the significance of these results in the physiology of respiration in Agaricus bisporus Lge are given.     

Immobilization of glycerol dehydrogenase on magnetic silica nanoparticles for conversion of glycerol to value-added 1,3-dihydroxyacetone

Abstract

Glycerol dehydrogenase (GlyDH) which oxidizes glycerol to the value-added chemical, 1,3-dihydroxyacetone, is of interest due to the oversupply of glycerol as a by-product of the biodiesel industry. To exploit the enzymatic oxidation of glycerol industrially, silica coated magnetic Fe₃O₄ nanoparticles were prepared and then activated with an amino-silane reagent for covalent immobilization of GlyDH via a glutaraldehyde linkage. At the optimal glutaraldehyde concentration of 0.05% (v/v), an enzyme loading of up to 57.5 mg/g-nanoparticles was achieved with 81.1% of the original activity retained. Reaction kinetic analysis indicated that the immobilized GlyDH had almost the same Michaelis-Menten constants for both NAD⁺ and glycerol as the free GlyDH did. However, after immobilization the turnover number k_cat of the GlyDH decreased from 164 s^?1 to 113 s^?1, and the reaction was 1.3-fold less sensitive to inhibition by DHA, which could compensate the decrease in k_cat. The immobilized GlyDH was also less sensitive to changes in pH and temperature, and showed a 5.3-fold improvement in thermal stability at 50^°C. Furthermore, excellent reusability was observed such that 10 cycles of re-use only led to 9% loss of enzyme activity.     

Immobilization of glucose isomerase to ion-exchange materials

Glucose isomerase (D -xylose ketol-isomerase EC 5.3.1.5) from Bacillus Coagulans was partially purified and immobilized by adsorption to anion exchangers. The highest activities were obtained when the enzyme was adsorbed to DEAE-cellulose. On immobilization to DEAE-cellulose the measured optimum pH value for enzyme activity shifted from 7.2 to 6.8. There was no appreciable difference between the heat stabilities of soluble and immobilized enzyme. The K_{m app} values for the immobilized enzyme were found to be 0.25M in the presence of 0.01M Mg²⁺ and 0.19M with 0.005M Mg²⁺, while those enzyme were 0.11 and 0.17M, re spectively. Under conditions of contimuous of D -glucose, a decrease of activity with time was observed, but this decrease was less at a low Mg²⁺ concentration and was affected by column geometry. There were no appreciable diffusional limitation effects in packed-bed columns.     

Purification and immobilization of NAD+ synthetase from Escherichia coli

The enzyme NAD⁺synthetase [deamido-NAD⁺: ammonia ligase (AMP-forming), EC 6.3.1.5] is used for the preparation of 2 μmol isotopically labelled [¹³N]NAD⁺, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high yield synthesis of [¹³N]NAD⁺, the NAD⁺synthetase is immobilized on porous glass beads and packed in a column. The NAD⁺synthetase was obtained from Escherichia coli. Different strains were tested; the cell culture technique was optimized. A new, high yield purification was applied. A screening of different immobilization techniques was done. The selected immobilization method was further optimized to increase the enzymatic activity of the enzyme-loaded glass beads. The latter were packed into a glass column. The kinetic properties of this column were investigated and optimized.     

Structural and functional characterization of BaiA,an enzyme involved in secondary bile acid synthesis in human gut microbe

Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo‐ and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2′‐phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (k_cat/K_M) of NADP⁺ at least an order of magnitude lower than NAD⁺. Substitution of Glu42 with Ala improved specificity toward NADP⁺ by 10‐fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide‐hydroxyl ion (NAD⁺‐OH^?) adduct contraposing previously reported adducts. The OH^? of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2′‐hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD⁺‐OH^? adduct in proton relay instead of hydride transfer as noted for previous adducts. Proteins 2014; 82:216–229. © 2013 Wiley Periodicals, Inc.     

Molecular dynamics simulation of the gGAPDH–NAD+ complex from Trypanosoma cruzi

A 50-ns molecular dynamics simulation has been used to study the homotetramer of the enzyme glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) complexes, from Trypanosoma cruzi, with nicotinamide adenine dinucleotide (NAD⁺) cofactors in aqueous solution. The root mean square deviation indicates that the overall structure of the homotetramer does not undergo significant change. The largest structural change observed was in the NAD⁺ binding domain of subunit (chain) D; as a consequence, the NAD⁺ cofactor was dislocated from its initial position. However, the other subunits were not affected, suggesting that the gGAPDH enzyme exhibits non-cooperative behaviour. Our simulation estimates that the NAD⁺ binding domain rotates about 4.8° relative to the catalytic domain in the apo–holo form transition. The hydrogen bond analysis reveals that the residues R12, I13, D38 and M39 are essential for gGAPDH–NAD⁺ interaction. Furthermore, two promising cavities to be explored in drug design were found: one formed by residues I13, R12, T197, T199, E336 and Y339, and the other by residues C166, H194, R249, I13, R12, T197, T199, E336 and Y339. The results presented in this paper offer new insight into the search for inhibitors of the gGAPDH enzyme of T. cruzi protozoan.     

Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.     

Xanthine dehydrogenase from Drosophila melanogaster: a comparison of the kinetic parameters of the pure enzyme from two wild-type isoalleles differing at a putative regulatory site.

Summary Xanthine dehydrogenase (XDH) from Drosophila melanogaster has been purified to homogeneity by immunoaffinity chromatography, and its kinetic parameters determined. Drosophila XDH exhibits ordered binding for substrate and NAD⁺, analogous to the corresponding enzymes from vertebrate sources. The wild-type enzyme exhibits a K_m for xanthine of 2.4x10^-5 M, and for NAD⁺ of 4.0x10^-5 M. XDH purified from a genetic variant exhibiting elevated levels of enzyme activity has similar kinetic constants. The results provide further evidence that the site of variation in the latter strain results in higher steady state numbers of XDH molecules per fly.     

NAD+ -dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD⁺-dependent and NADP⁺-dependent malic enzyme activities. The NAD⁺ malic enzyme exhibited more activity with NAD⁺ as cofactor, but also showed some activity with NADP⁺. The NADP⁺ malic enzyme only showed activity when NADP⁺ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NADP⁺ malic enzyme activity (dme) but retained NADP⁺ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N₂. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N₂-fixation would normally begin. These results support the proposal that NAD⁺ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N₂-fixing bacteroids which metabolize C₄-dicarboxylic acids supplied by the plant.     

Kinetics of a hollow fiber dehydrogenase reactor

A hollow fiber module was used as a reactor for conversion of ethanol to acetaldehyde in the presence of horse liver alcohol dehydrogenase as catalyst. Mass transport rates for NAD⁺, the overall acetaldehyde generation rate, catalyst effectiveness factors, and the overall order of the reaction with respect to NAD⁺ concentration were measured. A coupled-substrate reactor with continuous in situ regeneration of cofactor was also examined. Two substrates of opposite redox state were added simultaneously to the feed stream. NADH and acetaldehyde concentrations were monitored in the effluent stream. The cofactor recycle number, or ratio of moles of product to moles of NADH produced, exceeded 10,000 under certain conditions. While decreasing the NAD⁺ concentration in the feed stream decreased reactor productivity somewhat, it greatly enhanced the ratio of product formed per mole of NAD⁺ fed to the reactor. It is suggested that high cofactor costs in dehydrogenase reactors may be overcome with efficient in situ regeneration and secondary recovery and recycling of cofactor from the process stream.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Mechanism of Inhibition of the Human Sirtuin Enzyme SIRT3 by Nicotinamide: Computational and Experimental Studies

Sirtuins are key regulators of many cellular functions including cell growth, apoptosis, metabolism, and genetic control of age-related diseases. Sirtuins are themselves regulated by their cofactor nicotinamide adenine dinucleotide (NAD⁺) as well as their reaction product nicotinamide (NAM), the physiological concentrations of which vary during the process of aging. Nicotinamide inhibits sirtuins through the so-called base exchange pathway, wherein rebinding of the reaction product to the enzyme accelerates the reverse reaction. We investigated the mechanism of nicotinamide inhibition of human SIRT3, the major mitochondrial sirtuin deacetylase, in vitro and in silico using experimental kinetic analysis and Molecular Mechanics-Poisson Boltzmann/Generalized Born Surface Area (MM-PB(GB)SA) binding affinity calculations with molecular dynamics sampling. Through experimental kinetic studies, we demonstrate that NAM inhibition of SIRT3 involves apparent competition between the inhibitor and the enzyme cofactor NAD⁺, contrary to the traditional characterization of base exchange as noncompetitive inhibition. We report a model for base exchange inhibition that relates such kinetic properties to physicochemical properties, including the free energies of enzyme-ligand binding, and estimate the latter through the first reported computational binding affinity calculations for SIRT3:NAD⁺, SIRT3:NAM, and analogous complexes for Sir2. The computational results support our kinetic model, establishing foundations for quantitative modeling of NAD⁺/NAM regulation of mammalian sirtuins during aging and the computational design of sirtuin activators that operate through alleviation of base exchange inhibition.     

Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation,Kinetics and Evolutionary Studies

Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD⁺ and NADP⁺ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP⁺ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2''-phosphate of NADP⁺, but the same residues formed no observable interactions in the case of NAD⁺. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k_cat/K_M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP⁺, only R50 makes a contribution (about -1 kcal/mol) to NAD⁺ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP⁺ from NAD⁺. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP⁺-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD⁺ in the case of the NADP⁺-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.     

A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

The NAD⁺‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD⁺ and exhibits only a low activity with NADP⁺ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD⁺. While an application in the regeneration of NADP⁺ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP⁺‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP⁺ acceptance. The mutant V39R was identified to have increased activity with NADP⁺ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP⁺ in an enzyme‐coupled approach combined with the NADP⁺‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP⁺.     

A “Stripping” Ligand Tactic for Use with the Kinetic Locking-on Strategy: Its Use in the Resolution and Bioaffinity Chromatographic Purification of NAD-Dependent Dehydrogenases

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD⁺-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD⁺-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously “dribbled” off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5′-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD⁺-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27).     

Insights into the function of RifI2: Structural and biochemical investigation of a new shikimate dehydrogenase family protein

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD⁺ instead of NADP⁺ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD⁺ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD⁺-dependent member of the SDH family.