A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii.

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity.

Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of l-glutamate to α-ketoglutarate using NAD(P) as a cofactor. The bacterial enzymes are hexamers and each polypeptide consists of an N-terminal substrate-binding (Domain I) followed by a C-terminal cofactor-binding segment (Domain II). The reaction takes place at the junction of the two domains, which move as rigid bodies and are presumed to narrow the cleft during catalysis. Distinct signature sequences in the nucleotide-binding domain have been linked to NAD(+) vs. NADP(+) specificity, but they are not unambiguous predictors of cofactor preferences. Here, we have determined the crystal structure of NAD(+)-specific Peptoniphilus asaccharolyticus glutamate dehydrogenase in the apo state. The poor quality of native crystals was resolved by derivatization with selenomethionine, and the structure was solved by single-wavelength anomalous diffraction methods. The structure reveals an open catalytic cleft in the absence of substrate and cofactor. Modeling of NAD(+) in Domain II suggests that a hydrophobic pocket and polar residues contribute to nucleotide specificity. Mutagenesis and isothermal titration calorimetry studies of a critical glutamate at the P7 position of the core fingerprint confirms its role in NAD(+) binding. Finally, the cofactor binding site is compared with bacterial and mammalian enzymes to understand how the amino acid sequences and three-dimensional structures may distinguish between NAD(+) vs. NADP(+) recognition.

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined.

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-ß-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

Crystallization and preliminary structural analyses of glutamate dehydrogenase from Peptoniphilus asaccharolyticus.

Glutamate dehydrogenase (EC 1.4.1.2-4) from Peptoniphilus asaccharolyticus has been expressed as a selenomethionine-derivatized recombinant protein and diffraction-quality crystals have been grown that are suitable for structure determination. Preliminary structural analyses indicate that the protein assembles as a homohexameric enzyme complex in solution, similar to other bacterial and mammalian enzymes to which its sequence identity varies between 25 and 40%. The structure will provide insight into its preference for the cofactor NADH (over NADPH) by comparisons with the known structures of mammalian and bacterial enzymes.

Modular coenzyme specificity: a domain-swopped chimera of glutamate dehydrogenase.

Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD(+)-specific) and Escherichia coli (EcGDH) (NADP(+)-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain I) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain II; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain I. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher pH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V(max)) in both directions than either of the parent enzymes from which it is constructed.

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.

Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)(+) as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP(+) cofactor from the parent E. coli domain II, although there are subtle differences in catalytic activity.

Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

Structure of NADP(+)-dependent glutamate dehydrogenase from Escherichia coli--reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases.

Glutamate dehydrogenases (GDHs; EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD(+) and/or NADP(+) as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP(+) in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP(+) recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD(+)-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP(+). These studies clarify the sequence-structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

Reversal of the extreme coenzyme selectivity of Clostridium symbiosum glutamate dehydrogenase.

Active-site mutants of glutamate dehydrogenase from Clostridium symbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) with retention of high levels of catalytic activity for the new coenzyme. For oxidized coenzymes, k(cat) /K(m) ratios of the wild-type and triple mutant enzyme indicate a shift in preference of approximately 1.6 × 10(7) -fold, from ∼ 80,000-fold in favour of NAD(+) to ∼ 200-fold in favour of NADP(+). For reduced coenzymes the corresponding figure is 1.7 × 10(4) -fold, from ∼ 1000-fold in favour of NADH to ∼ 17-fold in favour of NADPH. A fourth mutation (N290G), previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant.