pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity.     

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an ‘ordered’ mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.     

Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention

The increasing availability of DNA-sequence information for multiple pathogenic and non-pathogenic variants of individual bacterial species has indicated that both DNA acquisition and genome reduction have important roles in genome evolution. Such genomic fluidity, which is found in human pathogens such as Escherichia coli, Helicobacter pylori and Mycobacterium tuberculosis, has important consequences for the clinical management of the diseases that are caused by these pathogens and for the development of diagnostics and new molecular epidemiological methods.     

Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.     

Cloning and deletion analysis of a genomic segment of Bacillus subtilis coding for the sdhA, B, C (succinate dehydrogenase) and gerE (spore germination) loci

From a Bacillus subtilis gene bank constructed in Escherichia coli and based on a low copy number cloning vector we have isolated a hybrid plasmid, pSH1047, containing an 8.0 kb segment of B. subtilis DNA coding for the sdhA, B and C genes, which code for the component polypeptides of succinate dehydrogenase, and the gerE gene, which may code for or regulate a protease involved in producing spores which germinate normally. We report the restriction map of this segment and the analysis of deletion derivatives which allow us to correlate the physical and genetic maps of these chromosomal segments.     

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O₂ and H₂O₂. SOD also reacts with H₂O₂, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO₂) and H₂O₂, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.