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The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes but still is a difficult synthetic challenge. Herein, we introduce natural hydroxyproline as a convertible unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, homoserine lactones, and Agl lactams, and to achieve the mild, efficient, and site-selective modification of peptides. A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both terminal and internal positions can be modified, and similar customizable units can be differentiated. The resulting products possess two reactive chains which can be manipulated independently. The versatility and scope of this process is highlighted by its application to the ligation of two peptide chains, and the generation of peptides with several chains and peptides with conformational restrictions. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) has recently been recognized as an important physiologically relevant gasotransmitter. Produced by the enzymes involved in the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), H2S has been implicated to control biological activity in virtually every organ system. In recent years it is being recognized that many commonly used H2S assays do not measure free H2S specifically and may be prone to artifacts. This has led to large variations in the reported H2S biological concentrations. In order to accurately study H2S's functions in biological systems accurate assays which measure free H2S specifically are required. In this work we present a simple microplate-based colorimetric assay for H2S gas. The underside of a 96-well microplate cover was coated with Nafion polymer doped with Ag+ ions. H2S is a highly volatile gas, and as it is volatilized in the microplate well it reacts with Ag+ to produce Ag2S nanoparticles, which have a strong absorbance in the low-UV range. By monitoring the absorbance change from formation of Ag2S nanoparticles, H2S production can be monitored in real time. The assay has a limit of detection (LOD) of 2.61 nmol (8.70 μM) and a liner range up to 30 nmol (100 μM). Using the assay, the KM and Vmax of recombinant CSE enzyme were determined to be 11.13 ± 0.57 mM and 0.45 ± 0.01 nmol min-1, respectively. H2S production from mouse liver homogenate under aerobic conditions in the presence of cysteine was measured and determined to be 4.89 ± 0.19 nmol min-1 mL-1 homogenate. The assay is simple, low cost, and specific to free H2S gas. [ABSTRACT FROM AUTHOR]

Cystathionine β-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (cCth) to produce c-homocysteuse, pyruvate, and ammonia. A series of site-directed vanants of Escherichia cub CBL (eCBL) was constructed to investigate the roles of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity. The eflëct of these conservative substitutions on the kmcat/Km L.Cth for the α-β-elimination of L-Cth ranges from a change of only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine replacement variant of S339. A novel role for residue S339 as a determinant of reaction specificity, via tethering of the catalytic base, K210, is demonstrated. Comparison of the kinetic parameters for i.-Cth hydrolysis with those for the inhibition of eCBL by aminoethoiyvinylglycine (AVG) indicates that Y238 interacts with the distal carboxylate group of the substrate. The 22 and SO-fold increases in the Kcat Km I.Cth and R ° resulting from replacement of Y56 with phenylalanine suggest that this residue may interact with the distal amino group of these compounds, although an indirect role in binding is more likely. The near-native kcat/KmI.Cth and pH profile of the eCBL-YIIIF variant demonstrate that residue Yl 11 does not play a role An proton transfer. The understanding of the eCBL active site and of the determinants of substrate and reaction specificity resulting from this work will facilitate the design of inhibitors, as antibacterial therapeutics, and the engineering of enzymes dependent on the catalytically versatile pyridosal S~-phosphate cofactor to modify reaction specificity. [ABSTRACT FROM AUTHOR]

A three-pot preparation of (S)-N-protected homoserine y-lactones is presented. Conversion of N-protected L-aspartic add to an oxazolidinone is followed by selective reduction/acid-catalyzed cydization to deliver the lactones. Microwave irradiation proved valuable for improving die latter reaction steps in some cases. [ABSTRACT FROM AUTHOR]

Human cystathionine β-synthase (hCBS), a key enzyme in the trans-sulfuration pathway, catalyzes the condensation of serine with homocysteine to produce cystathionine. CBS from higher organisms is the only known protein that binds pyridoxal-5′-phosphate (PLP) and heme. Intriguingly, the function of the heme in hCBS has yet to be elucidated. Herein, we describe the characterization of a cobalt-substituted variant of hCBS (Co hCBS) in which CoPPLX replaces FePPIX (heme). Co(III) hCBS is a unique Co-substituted heme protein the Co(III) ion is 6-coordinate, low-spin, diamagnetic, and bears a cysteine(thiolate) as one of its axial ligands. The peak positions and intensities of the electronic absorption and MCD spectra of Co(III) hCBS are distinct from those of previously Co-substituted heme proteins; TD-DFT calculations reveal that the unique features arise from the 6-coordinate Co bound axially by cysteine(thiolate) and a neutral donor, presumably histidine. Reactivity of Co(III) hCBS with HgCl2 is consistent with a loss of the cysteine(thiolate) ligand. Co(III) hCBS is slowly reduced to Co(II) hCBS, which contains a 5-coordinate, low-spin, S = 1/2 Co-porphyrin that does not retain the cysteine(thiolate) ligand; this form of Co(lI) hCBS binds NO(g) but not CO(g). Co(II) hCBS is reoxidized in the air to form a new Co(III) form, which does not contain a cysteine(thiolate) ligand. Canonical and altemative CBS assays suggest that maintaining the native heme ligation motif of wild-type Fe hCBS (Cys/His) is essential in maintaining maximal activity in Co hCBS. Correlation between the coordination structures and enzyme activity in both native Fe and Co-substituted proteins implicates a structural role for the heme in CBS. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals. i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H2S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457-22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H2S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H25 generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H2S and forms an aminoacrylate intermediate (kobs of 1.61 ± 0.04 mM-1 s-1 at low cysteine concentrations and 2.8 ± 0.1 mM-1 s-1 at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM-1 s-1 and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products. cystathionine and H2S. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by S-adenosyl- L-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conforrnational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chats reactivity of the truncated CBS lacking the regulatory domain (4SCBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms. [ABSTRACT FROM AUTHOR]

A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be involved in disulfide bonds. A differential alkylation method was developed to determine the percentage of free sulfhydryl at each cysteine residue of four recombinant monoclonal antibodies. Free sulfhydayl was first alkylated with 12C iodoacetic acid. Free sulfhydryl, resulting from the reduction of disulfide bonds, was then alkylated with 13C iodoacetic acid. Cysteine containing peptides that were modified by 13C iodoacetic acid showed a molecular weight that was 2 Da higher than the same peptide that was modified by 12C iodoacetic acid. Peptides, containing the same cysteine residues that were modified with both alkylation reagents, coeluted on reversed-phase chromatography. Analysis by mass spectrometry resulted in two partially overlapped m/z series for each cysteine containing peptide, corresponding to modification by iodoacetic acid with 12C or 13C. The percentage of free sulfhydryl was then calculated using the two mix series at each cysteine site. A low percentage of free sulfhydryl was detected at every cysteine residue in the four antibodies studied. Although different antibodies contained different levels of free sulfhydryl, similar distribution of free sulfhydryl in the domain structures was observed in the four antibodies. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'- phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C=N bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(ll)CBS, or by an Arg266Met replacement in Fe(lI)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring 03' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed. [ABSTRACT FROM AUTHOR]

The galacto-, homoserine-, mevalonate-, phosphonevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 A crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae complex with phosphomevalonate and AMPPNP·Mg2+. Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two antiparallel Iysines residues (100 and 101) with the result that Lys101 is switched into a position in which its ammonium ion is in direct contact with the β,γ-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary complex structures reveals that while their Cα-scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or nonreactive The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of the substrate Notably, a water pentamer that interacts extensively with the reactive group a of both substrates was discovered at the active tile. [ABSTRACT FROM AUTHOR]

Lactose repressor protein (LacI) utilizes an allosteric mechanism to regulate transcription in Escherichia coli, and the transition between inducer- and operator-bound states has been simulated by targeted molecular dynamics (TMD). The side chains of amino acids 149 and 193 interact and were predicted by TMD simulation to play a critical role in the early stages of the Lad conformational change. D149 contacts IPTG directly, and variations at this site provide the opportunity to dissect its role in inducer binding and signal transduction. Single mutants at D149 or S193 exhibit a minimal change in operator binding, and alterations in inducer binding parallel changes in operator release, indicating normal allosteric response. The observation that the double mutant D149A/S193A exhibits wild-type properties excludes the requirement for inter-residue hydrogen bond formation in the allosteric response. The double mutant D149C/S193C purified from cell extracts shows decreased sensitivity to inducer binding while retaining wild-type binding affinities and kinetic constants for both operator and inducer. By manipulating cystine oxidation, we show that the more reduced state of D149C/S193C responds to inducer more like the wild-type protein, whereas the more oxidized state displays diminished inducer sensitivity. These features of D149C/S193C indicate that the novel disulfide bond formed in this mutant impedes the allosteric transition, consistent with the role of this region predicted by TMD simulation. Together, these results establish the requirement for flexibility in the spatial relationship between D149 and S193 rather than a specific D149-S193 interaction in the Lad allosteric response to inducer. [ABSTRACT FROM AUTHOR]

Human cystathionine-γ-lyase (CGL) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c.1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in Vmax compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in Vmax. The KMs for cystathionine for both pathogenic, mutants are comparable to that of wild type CGL. The PLP content of" the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy. [ABSTRACT FROM AUTHOR]

The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series ofalanine mutants of the prototypic cyclotide kalata Bi were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In, this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 ± 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(Ill)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 ± 0.07) × 105 M-1 s-1 at pH 7.4 and 25 °C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 °C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely. [ABSTRACT FROM AUTHOR]

Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell-cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these ~90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium fume faciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses. [ABSTRACT FROM AUTHOR]

A modified LC-MS/MS method for large-scale screening of metabolic phenotypes was developed and validated. Twenty amino acids and 5 metabolically related compounds are measured within 4 min using multiple reaction monitoring (MRM) transitions selective for each compound. Separation with a short C18 column and rapid gradient using the ion-pairing reagent perfluoroheptanoic acid allows chromatographic resolution of the isomers Ile and Leu and improved chromatographic peak shapes for Lys, Arg, and His. MRM transitions were established with capability to distinguish isomers Leu from Ile and Thr from homoserine even when chromatographic resolution is incomplete. The reproducibility of the assay was tested by adding eight stable isotope-labeled amino acid standards (AA*) to extracts of Arabidopsis thaliana seeds. In intra- and interday assay comparisons, mean coefficients of variation of the peak area ratios of AA/AA* were less than 5% for all but Gly/Gly-D2-15N1. Recoveries of these eight amino acids ranged from 62 to 94% and suppression of signal by the matrix were 31-65%. Dilution of seed extracts reduced ion suppression for early-eluting amino acids but had minimal effects for those eluting later. The intra- and interday accuracies for these eight amino acids were 77-131 and 88-133% of nominal concentrations, respectively. [ABSTRACT FROM AUTHOR]

A novel DNA modification system by sulfur (S) in Streptomyces liuidans 66 was reported to be encoded by a cluster of five genes designated dndA-E [Zhou, X., He, X., Liang, J., Li, A., Xu, T., Kieser, T., Helmann, J. D., and Deng, Z. (2005) Mol. Microbiol. 57, 1428-1438]. The dndA gene was cloned and the protein product expressed in Escherichia coli, purified to homogeneity, and characterized as a homodimeric protein of ca. 91 kDa. Purified DndA has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate-containing enzyme and was proven to be a cysteine desulfurase able to catalyze removal of elemental S atoms from L-cysteine to produce L-alanine with substrate specificity similar to that of E. coli IscS. DndC was also purified to homogeneity and found to contain a 4Fe-4S cluster by spectral analysis and have obvious ATP pyrophosphatase activity. DndA could catalyze iron—sulfur cluster assembly by activation of apo-Fe DndC protein prepared by removal of its iron—sulfur cluster using α,α′-dipyridyl. A mutated DndA, with serine substituted for cysteine at position 327, which was confirmed to have lost its corresponding cysteine desulfurase activity, also lost its ability to reactivate the apo-Fe DndC. The likely involvement of an interaction between DndA and DndC in the biochemical pathway for the unusual site-specific DNA modification in S. lividans 66 is discussed. [ABSTRACT FROM AUTHOR]

Superoxide reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo- Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial FeCN and FeS bonding is probed by 13C ENDOR of the cyanide ligand and ¹Hβ ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the FeCN linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the spin density on the sulfur in both active sites to a degree that exceeds the difference induced by the alternative sets of ‘in-plane’ nitrogen ligands. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits the toxic metabolite, homocysteine, to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. The molecular basis of the organ- specific pathologies associated with CBS deficiency is unknown as is the significance of the reported interaction between CBS and Huntingtin protein. In this study, we have used the yeast two-hybrid approach to screen for proteins that interact with CBS and have identified several components of the sumoylation pathway including Ubc9, PIAS1, PIAS3, Pc2, and RanBPM. We demonstrate that CBS is modified by the small ubiquitin-like modifier-1 protein (SUMO-I) under both in vitro and in vivo conditions. Deletion analysis of CBS indicates that the C-terminal regulatory domain is required for interaction with proteins in the sumoylation machinery. Sumoylated CBS is present in the nucleus where it is associated with the nuclear scaffold. The discovery that CBS is a target of sumoylation adds another layer to the complex regulation of this enzyme and reveals a previously unknown residence for this protein, i.e., in the nucleus. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-ʟ-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand. [ABSTRACT FROM AUTHOR]

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of β sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 °C, while at HB positions there was a corresponding decrease of -7 °C. The reduced state of all proteins had similar stability. [ABSTRACT FROM AUTHOR]

Cyclic voltammetry of TEMPO in aqueous 0.1 M NaOH in the presence of glutathione (GSH) or cysteine (Cys) indicated the following points: (i) Both of the thiols rapidly formed adducts 3 with oxoammonium ion 1 anodically generated from TEMPO. (ii) 3 generated from GSH entered a succeeding reaction that generated N-oxide anion 2- (the reduced TEMPO). (iii) 3 produced from Cys remained intact over the time scale of voltammetry. A structural feature of GSH was considered to contribute to the observed behavior of this tripeptide. Possible structural features were evaluated by screening various thiols on the basis of whether they provided GSH-like voltammetric results. The 3-mercaptopropionamide group with an amide hydrogen in GSH was determined to be responsible for the observed difference between GSH and Cys. The likely function is to transform 3 from GSH into a 5-imino-1,2-oxathiolane intermediate, thereby releasing 2 . Product analysis for reactions of model thiols representing GSH and Cys with 1 provided support for this argument and suggested that the reaction of GSH or Cys with 1 would produce the corresponding disulfides, regardless of whether a five-membered ring intermediate was formed. The proposed function of the 3-mercaptopropionamide moiety of GSH may provide useful insight for the molecular design of exogenous thiol compounds as novel drugs for the treatment of GSH-depletion-related disorders. [ABSTRACT FROM AUTHOR]

A simple, rapid, rugged, sensitive, and specific method for the confirmation and quantitation of 10 β-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction, dispersive solid-phase extraction (dispersive- SPE) cleanup, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. Dispersive-SPE greatly simplifies and accelerates sample cleanup and improves overall recoveries com- pared with conventional SPE cleanup. The β-lactam antibiotics tested were as follows: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Average recoveries of fortified samples were 70% or better for all β-lactams except DCCD, which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service, which had previously tested the samples using the approved semiquantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method was superior in that it could specifically identify which β-lactam was present and quantitate its concentration, whereas the microbial assay could only identify the type of β-lactam present and report a concentration with respect to the microbial inhibition of a penicillin G standard. In addition, for 6 of the 23 samples, LC/MS/ MS analysis detected a penicillin and a cephalosporin β-lactam, whereas the microbial assay detected only a penicillin β-lactam. For samples that do not fall into the "general agreement" category, the most serious discrepancy involves two samples where the LC/MS/MS method detected a violative level of a cephalosporin β-lactam (deacetylcephapirin) in the first sample and a possibly violative level of desfuroylceftiofur in the second, whereas the microbial assay identified the two samples as having only violative levels of a penicillin β-lactam. [ABSTRACT FROM AUTHOR]

A [4Fe-4S] enzyme that decomposes L-cySteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 mm when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the α-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an α,β-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed. [ABSTRACT FROM AUTHOR]

Human cystathionine β-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated. [ABSTRACT FROM AUTHOR]

Cysteine desulfurases (CDs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that cleave sulfur from cysteine via an enzyme cysteinyl persulfide intermediate. In vitro studies of these enzymes have generally employed dithiothreitol as a cosubstrate to reductively cleave the persulfide intermediate, and it has been suggested that persulfide cleavage is the rate-limiting step for catalysis. In this study, the kinetics and mechanisms of cleavage of the persulfide intermediate in Slr0387 (CD-0387), a sequence group I (NifS/IscS-like) cysteine desulfurase from Synechocystis sp. PCC 6803, by physiological and nonphysiological reductants have been examined, and the extent to which this step is rate-limiting for catalysis has been determined. The observations that dithiols such as dithiothreitol (DTT) cleave the persulfide with ∼100-fold greater efficiency than structurally similar monothiols such as 2-mercaptoethanol (2-ME), that cleavage by DTT exhibits saturation kinetics, and that the dependence of the observed first- order rate constant for persulfide cleavage by DTT on the concentration of the dithiol corresponds precisely with that for formation of a complex between DTT and the PLP cofactor of the resting enzyme suggest that persulfide cleavage by dithiols occurs by prior formation of a complex, in which addition of one thiol to the cofactor positions the second thiol for attack. This conclusion and the observation that a second molecule of L-cysteine can bind to the cofactor in the persulfide form of CD-0387 explain why several CDs are subject to potent inhibition by L-cysteine during turnover with DTT: binding of L-cysteine prevents formation of the PLP-DTT adduct and renders the dithiol no better than a monothiol, which must react with the persulfide in bimolecular fashion. Consistent with this rationale, catalysis by CD- 0387 with 2-ME as cosubstrate, while less efficient, is not subject to potent inhibition by L-cysteine. The similarity of the maximum rate constant for persulfide cleavage by DTT to kcat suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that kcat is ∼10-fold greater when tris-(2-carboxyethyl)phosphine (TCEP), the most efficient persulfide cleaver identified, is used as the reducing cosubstrate. The faster turnover with TCEP provides a chemical model for activation of CD-0387 and other CDs by the presence of accessory factors that serve as efficient acceptors of the persulfide sulfur. [ABSTRACT FROM AUTHOR]

Cysteine desulfurases, designated NifS, IscS, and SufS, cleave L-cysteine to form alanine and an enzyme cysteinyl persulfide intermediate. Genetic studies on the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 have shown that of the three Nif/Isc/SufS-like proteins encoded in its genome only the sequence group II protein, Slr0077/SufS, is essential. This protein has been overexpressed in Escherichia coli, purified to homogeneity, shown to bind pyridoxal-5'-phosphate (PLP) and to catalyze cysteine desulfuration, and characterized in terms of its structure and kinetics. The results suggest that catalysis in the absence of accessory factors has two constituent pathways, one involving nucleophilic attack by C372 to form the Slr0077/SufS-bound cysteinyl persulfide intermediate and the second involving intermolecular attack by the sulfur of a second molecule of the substrate on the initial L-cysteine---PLP complex to form free L-cySteine persulfide. The second pathway is operant in the C372A variant protein, explaining why it retains significant activity, which is proportional to the concentration of L-cysteme (i.e., does not saturate). C-S bond cleavage by the first (normal) pathway is considerably less efficient than the equivalent step in a group I desulfurase (Slr0387) from the same organism (characterized in the accompanying paper). The 1.8 Å crystal structure of the protein, which is very similar to that previously reported for E. coli SufS, shows that the loop on which C372 resides is well-ordered and shorter by 11 residues than the corresponding disordered loop of the group I NifS-like protein from The rmotoga maritima. Sequence comparisons establish that the T. maritima and Slr0387 proteins have loops of similar length. The combined structural and kinetic data imply that the modest activity of Slr0077/SufS and other SufS proteins in comparison to their sequence group I (NifSfIscS-like) paralogues results from inefficiency in the nucleophilic attack step associated with differences in the structure or dynamics of this loop. The recent reports that SufS proteins can be activated manyfold by binding to Suffi thus implies that the accessory protein either accelerates nucleophilic attack by the conserved cysteine residue of SufS by a conformational mechanism or itself contributes a nucleophilic cysteine for more efficient intermolecular attack. [ABSTRACT FROM AUTHOR]

The long-term impacts of chronic metal exposure for aquatic biota are not well understood, partly due to a lack of understanding of metal specification within tissues. The objective of this study was to determine hepatic Zn speculation of rainbow trout (Oncorhnychus mykiss) exposed to Zn- enriched water in relation to unexposed (control) fish, through direct analysis of freeze-dried liver samples using synchrotron X-ray absorption spectroscopy (XAS). Juvenile rainbow trout (n=30) were exposed to Zn in a two-step process, 200 μg L-1 for 14 days, followed by 370 μg L-1 for 23 days. Thirty other trout were grown in a control treatment (10 μg Zn L-1). At the end of the experiment, three liver samples per treatment were collected, freezedried, ground, and mixed homogeneously. Although Zn concentration was higher in the Zn-exposed livers than in the control livers (22.32 vs 13.73 mg kg-1, respectively; p < 0.05), Zn speciation was similar for both groups. Extended X-ray absorption fine structure (EXAFS) spectroscopy indicated that Zn was coordinated to 4 sulfur atoms with an average Zn-S bond distance of 2.31 ± 0.02 Å. Sulfur K-XANES analysis confirmed that S was predominantly in reduced organic form analogous to cysteine. Our results are consistent with previous evidence for Zn(II) bonding to S in metallothionein proteins. These results suggest that the mechanisms for dealing with the extra load of bioaccumulated Zn in high exposure conditions were the same as in the control group. [ABSTRACT FROM AUTHOR]

Cystathionine γ-synthase (CGS) is a pyridoxal phosphate-dependent enzyme that catalyzes a γ-replacement reaction, in which the succinyl group of an O-succinyl-L-homoserine (L-OSHS) is displaced by the thiol of L-cysteine to form L-cystathionine, in the first step of the bacterial transsulfuration pathway. The mechanism of Escherichia coli CGS (eCGS) is ordered with L-OSHS associating before L-Cys (k[sub catR]/ K[sub Mr, sup L-OSHS] = 9.8 × 10[sup 4] M[sup -1] s[sup -1], where the subscript R denotes the replacement reaction). The mechanism becomes ping-pong (k[sub catR]/g[sub Mr, sup L-OSHS] = 4.9 × 10[sup 4] M[sup -1] s[sup -1]) at L-Cys concentrations lower than K[sub m, sup L-Cys]. The enzyme also catalyzes a competing γ-elimination reaction, in which L-OSHS is hydrolyzed to succinate, NH[sub 3], and α-ketobutyrate (k[sub catE]/K[sub Me, sup L-OSHS] = 1350 ± 90 M[sup -1] s[sup -1], where the subscript E denotes the elimination reaction). The k[sub cat]/K[sub m, sup L-OSHS] versus Ph profile of ECGS is bell-shaped for both reactions. The Ph optimum and the Pk[sub a] values for the acidic and basic limbs are 7.4, 6.8 ± 0.1, and 8.0 ± 0.1, respectively, for the elimination reaction and 7.8, 7.4 ± 0.1, and 8.3 ± 0.1, respectively, for the replacement reaction. The internal aldimine of ECGS remains protonated at Ph < 10.5, and the α-amino group of L-OSHS has a Pk[sub a] of 9.71 ± 0.01; therefore, neither limb of the k[sub cat]/K[sub m, sup L-OSHS] versus Ph profiles can be assigned to aldimine, or to L-OSHS prototropy. Novel continuous assays for the elimination reaction, employing D-2hydroxyisocaproate dehydrogenase, and for the substitution reaction, employing cystathionine β-lyase and L-lactate dehydrogenase as coupling enzymes, are described. [ABSTRACT FROM AUTHOR]

An efficient method for data processing and interpretation is needed to support and extend disulfide mass-mapping methodology based on partial reduction and cyanylation-induced cleavage to proteins containing more than four cystines. Here, the concept of “negative signature mass” is introduced as the novel feature of an algorithm designed to identify the disulfide structure of a cystinyl protein given an input of mass spectral data and an amino acid sequence. The “negative signature mass” process is different from the conventional approach in that it does not directly rule-in disulfide linkages, but rather eliminates linkages from a list of all possible theoretical linkages, with the goal of ruling out enough linkages so that only one disulfide structure can be constructed. The operating principles and the effectiveness of the algorithm are described in the context of analyzing ribonuclease A, a 124-residue protein containing eight cysteines in the form of four cystines (disulfides). [Copyright &y& Elsevier]

Cystathionine β-synthase (CBS) is a pyridoxal-phosphate-dependent enzyme that catalyzes a β-replacement reaction in which the hydroxyl group of serine (L-Ser) is displaced by the thiol of homocysteine (L-Hcys) to form cystathionine (L-Cth) in the first step of the trans-sulfuration pathway. A new continuous assay for the forward reaction, employing cystathionine β-lyase and L-lactate dehydrogenase as coupling enzymes, is described. It alleviates product inhibition by L-Cth and revealed that the values for K[sup L-Ser, sub m] (1.2 mM) and for substrate inhibition by L-Hcys (K[sup L-Hcys, sub iF1] = 2.0 mM) are lower than those previously reported. A continuous, 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB)-based assay for the CBScatalyzed hydrolysis of L-Cth to L-Ser and L-Hcys provides a tool for investigation of the reverse reaction (k[sub catR] = 0.56 s[sup -1], K[sup L-Cth, sub m] = 0.083 mM). The k[sub catR]/K[sup L-Cth, sub m] versus pH profile of ytCBS is bell-shaped with a pH optimum of 8.3, and the pK[sub a] values for the acidic and basic limbs are 8.05 and 8.63, respectively. The latter is assigned to the α-amino group of L-Cth (pK[sub a] = 8.54). The internal aldimine of ytCBS remains protonated at pH < 11; therefore, the acidic pK[sub a] is assigned to an enzyme functionality that is not associated with the internal aldimine. K[sub eq] was determined directly and from the kinetic parameters, and the values are 0.61 and 1.2 µM, respectively. [ABSTRACT FROM AUTHOR]

Examines the role of disulfide bonds in folding activity of leirotoxin I. Formation of helix by three disulfide bridges; Reduction on biological potency; Modification of conserved cysteine residues affecting folding efficiency and structure.

Examines the functionality of cytochrome c[sub 1] redox potentials in the Rhodobacter capsulatus b[sub 1] complex. Number of cysteines contained in cytochrome c[sub 1]; Correlation between the opening of the heme and the drop in potential; Integrity of the heme-binding pocket.

Examines the reaction mechanism of cystathionine beta-synthase that forms a series of intermediates in the conversion of aminoacrylate to cystathionine. Use of rapid-kinetic and steady-state spectroscopy; Analysis of cystathionine beta-synthase reaction kinetics; Results of steady-state spectroscopic studies.

Determines the catalytic mechanism and substrate specificity of homoserine kinase. Identification on structures of homoserine kinase; Determination of homoserine binding site; Changes around the active site.

Examines the kinetics in the pre-steady state of the formation of cystines in ribonucleoside diphosphate reductase. Determination on the percent of the peripheral cystines; Formation of the cystine between cysteine 225 and 462; Evidence for an asymmetric complex.

Examines the kinetic, spectroscopic and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase. Purification of truncated form of cystathionine beta-synthase lacking the C-terminal 143 residue; Iron stoichiometry of the dimeric enzyme; Functions of the different regions of the cystathionine beta-synthase polypeptide.

Presents a study about the mutation to a serine residue of cysteines that comprise the active site disulfide in lipoamide dehydrogenase, to give the altered enzymes, C44S and C495. Redox behavior of the FAD in the absence of disulfide; Cause of the changes in slope at pH 7.9; Effect on redox of pyridine nucleotide binding.

Presents the procedure to determine the relative positions of a reactive cysteine residue on the gamma subunit of the retinal cyclic GMP phosphodiesterase and a reactive lysine residue on the alpha subunit of transducin. Introduction; Materials used and methodology; Results; Discussion.

Due to its unique flavor profile, Lentinula edodes has become one of the most popular edible mushrooms in the world, but the regulatory mechanism of its flavor substances has not been revealed. To study the mechanism that regulates the anabolic metabolism of the important flavor substance lenthionine (LT), the effect of cysteine (Cys) synthesized by the cystathionine-γ-lyase ( CSE-1 ) gene participating in the regulation of LT metabolism under drought stress was analyzed. Our results showed that drought stress promoted the accumulation of LT, and the key genes GTT and LECSL were activated. Furthermore, drought stress promoted the accumulation of intracellular Cys and activated the key gene for Cys synthesis, CSE-1 . Both inhibition of the CSE enzyme activity by inhibitors and silencing of the CSE-1 gene under drought stress significantly reduced the intracellular contents of Cys and LT, but the inhibition of LT synthesis disappeared after the exogenous addition of Cys. These results indicate that LT synthesis in L. edodes under drought stress is dependent on Cys. In summary, the mechanism of the regulation of flavor substances in edible mushrooms by the environment was revealed for the first time.

Methylmercury (MeHg) covalently modifies cellular proteins through their SH groups, resulting in cytotoxicity. We report that cystathionine β-synthase (CBS), which catalyzes the production of hydrogen sulfide, contributes to cellular protection against MeHg. Pretreatment with NaHS or overexpression of CBS reduced MeHg cytotoxicity, whereas transfection with CBS small interfering RNA enhanced MeHg toxicity in human neuroblastoma SH-SY5Y cells. Bismethylmercury sulfide ((MeHg)2S) was identified as a metabolite of MeHg in SH-SY5Y cells exposed to MeHg and in the livers of rats treated with MeHg. (MeHg)2S had little chemical protein modification capability and little cytotoxicity compared with MeHg in vitroand in vivo. [ABSTRACT FROM AUTHOR]

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H2S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H2S, via heme-mediated allosteric regulation of CBS. [ABSTRACT FROM AUTHOR]

The preparation and physical characterization are reported for a novel single-molecule magnet [Mn12O12(Oac)12(dpp)4] (dppH = diphenyl phosphate) with no coordinating water molecules. The crystal structure analysis reveals that there are four five-coordinate MnIII ions with Mn…H approaches. Addition of water in CD2Cl2 solution was monitored by 1H NMR, which showed that H2O could coordinate to a vacant site of a five-coordinate MnIII ion in solution. The measurements and analyses of magnetization hysteresis and ac magnetic susceptibility indicate that the title complex is a single-molecule magnet with a quantum tunneling behavior, whose ground state was tentatively assigned to S = 10 with g = 1.78 and D = -0.60 K. [ABSTRACT FROM AUTHOR]

Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.