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.

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.

Summary Glutamate dehydrogenases (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 glutamate dehydrogenase (GDH) family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5Å 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 residue that follows the P7 aspartate is presumed to form a hydrogen bond to 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 toward the co-ordination of the 2’-ribose phosphate of NADP+. These studies clarify the sequence/structure relationships in bacterial glutamate dehydrogenases, 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.

The synthesis of N-heterocyclic carbene (NHC) silver(I) acetate complexes with varying lipophilic benzyl-substituents at the 1 and 3 positions starting from 4,5-diphenylimidazole, opened a new class of antibiotic drug candidates. These NHC-silver(I) acetate derivatives exhibit interesting structural motifs in the solid state and proved to be soluble and stable in biological media. The leading candidate, SBC3, which was known to exhibit good antibacterial activity in preliminary Kirby-Bauer tests, was tested quantitatively using minimum inhibitory concentrations. NHC-silver(I) acetate complexes were found to have MIC values ranging from 20 to 3.13 μg/mL for a variety of Gram-positive, Gram-negative and mycobacteria tested. These values represent good antibiotic activities against potential pathogens when compared to clinically approved antibiotics. Most striking is the fact that SBC3 is active against methicillin-resistant Staphylococcus aureus with a MIC value of 12.5 μg/mL.

No abstract