Cold-adapted alanine dehydrogenases from two antarctic bacterial strains: gene cloning, protein characterization, and comparison with mesophilic and thermophilic counterparts.
The genes encoding NAD(+)-dependent alanine dehydrogenases (AlaDHs) (EC 18.104.22.168) from the Antarctic bacterial organisms Shewanella sp. strain Ac10 (SheAlaDH) and Carnobacterium sp. strain St2 (CarAlaDH) were cloned and expressed in Escherichia coli. Of all of the AlaDHs that have been sequenced, SheAlaDH exhibited the highest level of sequence similarity to the AlaDH from the gram-negative bacterium Vibrio proteolyticus (VprAlaDH). CarAlaDH was most similar to AlaDHs from mesophilic and thermophilic Bacillus strains. SheAlaDH and CarAlaDH had features typical of cold-adapted enzymes; both the optimal temperature for catalytic activity and the temperature limit for retaining thermostability were lower than the values obtained for the mesophilic counterparts. The k(cat)/K(m) value for the SheAlaDH reaction was about three times higher than the k(cat)/K(m) value for VprAlaDH, but it was much lower than the k(cat)/K(m) value for the AlaDH from Bacillus subtilis. Homology-based structural models of various AlaDHs, including the two psychotropic AlaDHs, were constructed. The thermal instability of SheAlaDH and CarAlaDH may result from relatively low numbers of salt bridges in these proteins. (+info)
Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase.
The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M. tuberculosis, but not in the vaccine strain Mycobacterium bovis BCG. It is a functional L-alanine dehydrogenase (EC 22.214.171.124) and hence one of the few antigens possessing an enzymic activity. This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions. Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme. In the oxidative-deamination reaction, the enzyme showed K(m) values of 13. 8 mM and 0.31 mM for L-alanine and NAD(+), respectively, in a random-ordered mechanism. K(m, app) values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 microM for ammonium, pyruvate and NADH, respectively. The enzyme is highly specific for all of its substrates in both directions. The pH profile indicates that oxidative deamination virtually may not occur at physiological pH. Hence L-alanine most likely is the product of the reaction catalysed in vivo. The enzyme is heat-stable, losing practically no activity at 60 degrees C for several hours. (+info)
Structure and mechanism of proton-translocating transhydrogenase.
Recent developments have led to advances in our understanding of the structure and mechanism of action of proton-translocating (or AB) transhydrogenase. There is (a) a high-resolution crystal structure, and an NMR structure, of the NADP(H)-binding component (dIII), (b) a homology-based model of the NAD(H)-binding component (dI) and (c) an emerging consensus on the position of the transmembrane helices (in dII). The crystal structure of dIII, in particular, provides new insights into the mechanism by which the energy released in proton translocation across the membrane is coupled to changes in the binding affinities of NADP(+) and NADPH that drive the chemical reaction. (+info)
Disruption of aldA influences the developmental process in Myxococcus xanthus.
Previously, we identified a gene (aldA) from Myxococcus xanthus, which we suggested encoded the enzyme alanine dehydrogenase on the basis of similarity to known Ald protein sequences (M. J. Ward, H. Lew, A. Treuner-Lange, and D. R. Zusman, J. Bacteriol. 180:5668-5675, 1998). In this study, we have confirmed that aldA does encode a functional alanine dehydrogenase, since it catalyzes the reversible conversion of alanine to pyruvate and ammonia. Whereas an aldA gene disruption mutation did not significantly influence the rate of growth or spreading on a rich medium, AldA was required for growth on a minimal medium containing L-alanine as the major source of carbon. Under developmental conditions, the aldA mutation caused delayed aggregation in both wild-type (DZ2) and FB (DZF1) strains. Poorly formed aggregates and reduced levels of spores were apparent in the DZ2 aldA mutant, even after prolonged development. (+info)
Protein-coding genes as molecular markers for ecologically distinct populations: the case of two Bacillus species.
Bacillus globisporus and Bacillus psychrophilus are one among many pairs of ecologically distinct taxa that are distinguished by very few nucleotide differences in 16S rRNA gene sequence. This study has investigated whether the lack of divergence in 16S rRNA between such species stems from the unusually slow rate of evolution of this molecule, or whether other factors might be preventing neutral sequence divergence at 16S rRNA as well as every other gene. B. globisporus and B. psychrophilus were each surveyed for restriction-site variation in two protein-coding genes. These species were easily distinguished as separate DNA sequence clusters for each gene. The limited ability of 16S rRNA to distinguish these species is therefore a consequence of the extremely slow rate of 16S rRNA evolution. The present results, and previous results involving two Mycobacterium species, demonstrate that there exist closely related species which have diverged long enough to have formed clearly separate sequence clusters for protein-coding genes, but not for 16S rRNA. These results support an earlier argument that sequence clustering in protein-coding genes could be a primary criterion for discovering and identifying ecologically distinct groups, and classifying them as separate species. (+info)
Reassessment of major products of N2 fixation by bacteroids from soybean root nodules.
NH3/ was the principal product from soybean bacteroids, prepared by various procedures, when assayed in solution in a flow chamber under N2 fixation conditions. In addition, small quantities of alanine were produced (reaching 20% of NH3/ under some conditions). Some 15N was assimilated by bacteroids purified from soybean root nodules on Percoll density gradients and shaken with 15N2 and 0.008 atm O2. Under these conditions, accounted for 93% of the (15)N fixed into the soluble fraction. This fraction contained no measurable [15N]alanine. Neither these bacteroids nor those prepared by the previously used differential centrifugation method, when incubated with exogenous alanine under non-N2-fixing conditions, gave rise to NH3 from alanine. Therefore, contamination of bacteroid preparations with enzymes of plant cytosolic origin and capable of producing NH3 from alanine cannot explain the failure to detect [15N]alanine [as reported elsewhere: Waters, J. K., Hughes, B. L., II, Purcell, L. C., Gerhardt, K. O., Mawhinney, T. P. & Emerich, D. W. (1998). Proc Natl Acad Sci USA 95, 12038-12042]. Cell-free extracts of the bacteroids as used in the 15N experiments contained alanine dehydrogenase and were able to produce alanine from pyruvate and. Other experiments with alanine dehydrogenase in extracts of cultured rhizobia and bacteroids are reported and discussed in relation to the 15N experiments. Possible reasons for the differences between laboratories regarding the role of alanine are discussed. It is concluded that NH3 is the principal soluble product of N2 fixation by suspensions of soybean bacteroids ex planta and that should continue to be considered the principal product of N2 fixation which is assimilated in vivo in soybean nodules. (+info)
Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins.
The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli. (+info)
Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth.
NAD(H)-dependent L-alanine dehydrogenase (EC 126.96.36.199) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 188.8.131.52) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth. (+info)