Nitric oxide reversibly inhibits Bacillus subtilis oxalate decarboxylase.
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ABSTRACT:
SUBMITTER: Moral ME
PROVIDER: S-EPMC3057162 | biostudies-literature | 2011 Mar
REPOSITORIES: biostudies-literature
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Nitric oxide reversibly inhibits Bacillus subtilis oxalate decarboxylase.
Moral Mario E G ME Tu Chingkuang C Imaram Witcha W Angerhofer Alexander A Silverman David N DN Richards Nigel G J NG
Chemical communications (Cambridge, England) 20110124 11
Membrane inlet mass spectrometry (MIMS) has been employed to assay the catalytic activity of oxalate decarboxylase (OxDC), allowing us to demonstrate that nitric oxide (NO) reversibly inhibits the enzyme under dioxygen-depleted conditions. X-band EPR measurements do not provide any direct evidence for the interaction of NO with either of the Mn(II) centers in OxDC raising the possibility that there is a separate dioxygen-binding pocket in the enzyme. ...[more]
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Project description:Oxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn-dependent hexameric enzyme that converts oxalate to carbon dioxide and formate. OxDC has greatly attracted the interest of the scientific community, mainly due to its biotechnological and medical applications in particular for the treatment of hyperoxaluria, a group of pathologic conditions caused by oxalate accumulation. The enzyme has an acidic optimum pH, but most of its applications involve processes occurring at neutral pH. Nevertheless, a detailed biochemical characterization of the enzyme at neutral pH is lacking. Here, we compared the structural-functional properties at acidic and neutral pH of wild-type OxDC and of a mutant form, called OxDC-DSSN, bearing four amino acid substitutions in the lid (Ser161-to-Asp, Glu162-to-Ser, Asn163-toSer, and Ser164-to-Asn) that improve the oxalate oxidase activity and almost abolish the decarboxylase activity. We found that both enzymatic forms do not undergo major structural changes as a function of pH, although OxDC-DSSN displays an increased tendency to aggregation, which is counteracted by the presence of an active-site ligand. Notably, OxDC and OxDC-DSSN at pH 7.2 retain 7 and 15% activity, respectively, which is sufficient to degrade oxalate in a cellular model of primary hyperoxaluria type I, a rare inherited disease caused by excessive endogenous oxalate production. The significance of the data in the light of the possible use of OxDC as biological drug is discussed. © 2019 IUBMB Life, 1-11, 2019.
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Project description:Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).
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| S-EPMC2041844 | biostudies-literature
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| S-EPMC2688449 | biostudies-literature
Project description:YvrI is a recently identified alternative sigma factor in Bacillus subtilis that requires the coactivator YvrHa to activate transcription. Previously, a strain engineered to overproduce YvrI was found to overproduce oxalate decarboxylase (OxdC), and further analysis identified three YvrI-activated promoters preceding the yvrI-yvrHa, yvrJ, and oxdC-yvrL operons. Independently, proteome analyses identified OxdC as a highly abundant, cell wall-associated protein that accumulated under acidic growth conditions. We show here that the accumulation of OxdC in the cell wall proteome under acidic growth conditions is absolutely dependent on YvrI and is correlated with enhanced transcription of both the yvrI-yvrHa and the oxdC-yvrL operons. Conversely, OxdC accumulates to a high level even under nonacidic growth conditions in cells lacking YvrL, a negative regulator of YvrI/YvrHa-dependent transcription. These results indicate that YvrI and its associated coregulators YvrHa and YvrL are required for the regulation of OxdC expression by acid stress. The high-level accumulation of OxdC depends, in part, on a strong oxdC promoter. A regulatory sequence with similarity to an upstream promoter element (UP) was identified upstream of the oxdC promoter and is required for high-level promoter activity. Conservation of the YvrI/YvrHa/YvrL regulatory system among related species allowed us to deduce an expanded consensus sequence for the compositionally unusual promoters recognized by this new sigma factor.
| S-EPMC2632099 | biostudies-literature
Project description:Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl}propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor.
| S-EPMC1218680 | biostudies-other
Project description:We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.
| S-EPMC438601 | biostudies-literature
Project description:BackgroundBacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610.ResultsWe show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal.ConclusionThe results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.
| S-EPMC3224222 | biostudies-literature