Effect of hPc2 on sumoylation of human CBS.

<p>A. The in vitro sumoylation reaction was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004032#s4" target="_blank">Methods</a> and SUMO-1 antibody was used to detect the presence of sumoylated CBS. Lanes 1 and 2 show sumoylation reactions that were conducted in the absence (lane 1) and presence (lane 2) of hPc2. B and C. Equal loading controls for CBS in the two reaction mixtures. The CBS concentration prior to the start of the sumoylation reaction was identical in both lanes as detected by Coomassie blue staining (B) or by Western blot analysis using CBS antibody (C).</p

Effect of the H₂S generating substrates, cysteine and homocysteine, and the CBS reaction product, cystathionine, on sumoylation in the presence and absence of hPc2.

<p>The Western blot was detected using SUMO-1 antibody.</p

Structures of human CBS and CSE showing locations of potential SUMO modification sites.

<p>A. Locations of K211 and K269 in the canonical and noncanonical SUMO modification sites respectively in human CBS (PDB 1M54). The two subunits of CBS are shown in grey and blue and the heme and PLP cofactors are shown in red and yellow respectively. K211 and K269 are shown in one of the two subunits in navy and cyan respectively. B. Locations of K361 (blue), K330 (cyan) and K260 (red) in one of the four subunits of human CSE (PDB file 2NMP). The PLP cofactor is shown in yellow in ball and stick representation.</p

Effect of substrates on the sumoylation of human CBS.

<p>Western blot analysis using CBS-antibody of sumoylation reaction mixtures containing the substrates for CBS in the absence (A) or presence (B) of the allosteric activator, S-adenosylmethionine (AdoMet). (C) The effect of hPc2 on sumoylation of human CBS in the presence of substrates.</p

Human CSE is a target of in vitro sumoylation.

<p>Sumoylation of CSE was conducted as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004032#s4" target="_blank">Methods</a> in the presence or absence of 5 mM cystathionine. The Western blot was detected using SUMO antibody. The band with a molecular mass of ∼16 kDa represents SUMO-1 present in the reaction mixture.</p

Unusual Aerobic Stabilization of Cob(I)alamin by a B₁₂-Trafficking Protein Allows Chemoenzymatic Synthesis of Organocobalamins

CblC, a B₁₂ trafficking protein, exhibits glutathione transferase and reductive decyanase activities for processing alkylcobalamins and cyanocobalamin, respectively, to a common intermediate that is subsequently converted to the biologically active forms of the cofactor. We recently discovered that the Caenorhabditis elegans CblC catalyzes thiol-dependent decyanation of CNCbl and reduction of OH₂Cbl and stabilizes the paramagnetic cob­(II)­alamin product under aerobic conditions. In this study, we report the striking ability of the worm CblC to stabilize the highly reactive cob­(I)­alamin product of the glutathione transferase reaction. The unprecedented stabilization of the supernucleophilic cob­(I)­alamin species under aerobic conditions by the intrinsic thiol oxidase activity of CblC, was exploited for the chemoenzymatic synthesis of organocobalamin derivatives under mild conditions

Modulation of Catalytic Promiscuity during Hydrogen Sulfide Oxidation

The mitochondrial sulfide oxidation pathway prevents the toxic accumulation of hydrogen sulfide (H₂S), a signaling molecule that is maintained at low steady-state concentrations. Sulfide quinone oxidoreductase (SQR), an inner mitochondrial membrane-anchored protein, catalyzes the first and committing step in this pathway, oxidizing H₂S to persulfide. The catalytic cycle comprises sulfide addition to the active site cysteine disulfide in SQR followed by sulfur transfer to a small molecule acceptor, while a pair of electrons moves from sulfide, to FAD, to coenzyme Q. While its ability to oxidize H₂S is well characterized, SQR exhibits a remarkable degree of substrate promiscuity <i>in vitro</i> that could undermine its canonical enzyme activity. To assess how its promiscuity might be contained <i>in vivo</i>, we have used spectroscopic and kinetic analyses to characterize the reactivity of alternate substrates with SQR embedded in nanodiscs (<i>nd</i>SQR) versus detergent-solubilized enzyme (<i>s</i>SQR). We find that the membrane environment of <i>nd</i>SQR suppresses the unwanted addition of GSH but enhances sulfite addition, which might become significant under pathological conditions characterized by elevated sulfite levels. We demonstrate that methanethiol, a toxic sulfur compound produced in significant quantities by colonic and oral microbiota, can add to the SQR cysteine disulfide and also serve as a sulfur acceptor, potentially interfering with sulfide oxidation when its concentrations are elevated. These studies demonstrate that the membrane environment and substrate availability combine to minimize promiscuous reactions that would otherwise disrupt sulfide homeostasis

Reversible generation and metabolic consequences of Fe^II-NO CBS.

<p>(<b>A</b>) Reduction of Fe^III-CBS (10 µM) in 0.1 M HEPES buffer, pH 7.4, (-••-) with dithionite (3 mM) yields Fe^II-CBS (–). The latter reacts with 10 mM nitrite to give Fe^II-NO CBS (^….). The NO ligand is exchanged for CO upon incubation of the reaction mixture for 10–15 min with CO (––). (<b>B</b>) Effect of NO binding to Fe^II-CBS on H₂S production was measured in 0.1 M HEPES buffer, pH 7.4 using cysteine (10 mM) and homocysteine (10 mM) as substrates. H₂S generation was assesed using the lead sulfide precipitation assay. (<b>C</b>) Predicted metabolic consequences of Fe^II-NO CBS formation. Inhibition of CBS by its nitrite reductase activity is predicted to decrease CBS-dependent H₂S formation while increasing cystathionase (CSE)-dependent H₂S formation due to homocysteine accumulation. The concentration of the antioxidant glutathione (GSH), is also predicted to decrease.</p

Nitrite reduction by Fe^II-CBS.

<p>(<b>A</b>) UV-visible spectra recorded every minute under anaerobic conditions for the reaction between Fe^II-CBS (10 µM, generated by reduction of Fe^III-CBS with 3 mM dithionite) and nitrite (10 mM) in 0.1 M HEPES buffer, pH 7.4, at 37°C. (<i>Inset</i>) The observed reaction rate for CBS-catalyzed reaction as a function of nitrite concentration. (<b>B</b>) The disappearance of Fe^II-CBS (formed by reduction of Fe^III-CBS (10 µM) with dithionite (3 mM)) was monitored at 449 nm (filled circles) and paralleled the formation of Fe^II-NO-CBS in the presence of 10 mM nitrite (open circles) monitored at 394 nm. The solid lines represent single exponential fits to the experimental data points. (<b>C</b>) Dependence of nitrite reduction by Fe^II-CBS on pH. Reaction of Fe^II-CBS (10 µM) generated by the reduction of Fe^III-CBS with dithionite (3 mM) in 0.1 M HEPES pH 7.0, 7.25, 7.4, 7.75 and 8.0 at 37°C with nitrite (10 mM) was monitored at 449 nm. Reaction rates corrected for the percentage of reduced protein at each pH were plotted as a function of pH. The slope obtained from a linear fit was 1.2±0.03.</p

Model for and spectroscopic evidence of formation of Fe^II-NO CBS in the presence of MSR/NADPH.

<p>(<b>A</b>) Fe^III-CBS catalyzes the condensation of cysteine (Cys) and homocysteine (Hcy) to give H₂S and cystathionine (Cyst). The latter is subsequently cleaved to give cysteine, which is utilized for glutathione (GSH) synthesis. In the presence of NADPH/MSR and nitrite, Fe^II-NO CBS is formed, rendering CBS inactive. (<b>B</b>) EPR spectra of Fe^II-NO CBS, obtained with Fe^III-CBS (65 µM), treated with dithionite (6 mM) (upper) or NADPH (2 mM)/MSR (20 µM) (lower) and sodium nitrite (10 mM) in 0.1 M HEPES buffer, pH 7.4 at 37°C. The spectra were recorded using the conditions described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085544#pone.0085544-Taoka3" target="_blank">[26]</a>. The arrows indicate <i>g</i> values of 2.17, 2.076, 2.008 and 1.97, respectively. The presence of additional EPR signals in the spectrum of NADPH/MSR-dependent CBS-catalyzed nitrite reduction can be attributed to the incomplete reduction of paramagnetic Fe^III-CBS. (C) UV-visible spectra were recorded every 10 min under anaerobic conditions for the reaction between Fe^II-CBS (generated by reduction of Fe^III-CBS (10 µM) with MSR (2 µM)/NADPH (1 mM)) and nitrite (10 mM) in 0.1 M HEPES buffer, pH 7.4, at 37°C. (B) Time-dependent conversion of Fe^III-CBS (429 nm) to Fe^II-NO-CBS (394 nm).</p