New insights into immunomodulation via overexpressing lipoic acid synthase as a therapeutic potential to reduce atherosclerosis.

Atherosclerosis is a systemic chronic inflammatory disease. Many antioxidants including alpha-lipoic acid (LA), a product of lipoic acid synthase (Lias), have proven to be effective for treatment of this disease. However, the question remains whether LA regulates the immune response as a protective mechanism against atherosclerosis. We initially investigated whether enhanced endogenous antioxidant can retard the development of atherosclerosis via immunomodulation. To explore the impact of enhanced endogenous antioxidant on the retardation of atherosclerosis via immune regulation, our laboratory has recently created a double mutant mouse model, using apolipoprotein E-deficient (Apoe-/-) mice crossbred with mice overexpressing lipoic acid synthase gene (LiasH/H), designated as LiasH/HApoe-/- mice. Their littermates, Lias+/+Apoe-/- mice, served as a control. Distinct redox environments between the two strains of mice have been established and they can be used to facilitate identification of antioxidant targets in the immune response. At 6 months of age, LiasH/HApoe-/- mice had profoundly decreased atherosclerotic lesion size in the aortic sinus compared to their Lias+/+Apoe-/- littermates, accompanied by significantly enhanced numbers of regulatory T cells (Tregs) and anti-oxidized LDL autoantibody in the vascular system, and reduced T cell infiltrates in aortic walls. Our results represent a novel exploration into an environment with increased endogenous antioxidant and its ability to alleviate atherosclerosis, likely through regulation of the immune response. These outcomes shed light on a new therapeutic strategy using antioxidants to lessen atherosclerosis.

Biosynthesis of Sulfur-Containing Small Biomolecules in Plants.

Sulfur is an essential element required for plant growth. It can be found as a thiol group of proteins or non-protein molecules, and as various sulfur-containing small biomolecules, including iron-sulfur (Fe/S) clusters, molybdenum cofactor (Moco), and sulfur-modified nucleotides. Thiol-mediated redox regulation has been well investigated, whereas biosynthesis pathways of the sulfur-containing small biomolecules have not yet been clearly described. In order to understand overall sulfur transfer processes in plant cells, it is important to elucidate the relationships among various sulfur delivery pathways as well as to investigate their interactions. In this review, we summarize the information from recent studies on the biosynthesis pathways of several sulfur-containing small biomolecules and the proteins participating in these processes. In addition, we show characteristic features of gene expression in Arabidopsis at the early stage of sulfate depletion from the medium, and we provide insights into sulfur transfer processes in plant cells.

Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants.

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.

Effect of S-Allyl -L-Cysteine on MCF-7 Cell Line 3-Mercaptopyruvate Sulfurtransferase/Sulfane Sulfur System, Viability and Apoptosis.

The S-Allyl-L-cysteine ​​(SAC) component of aged garlic extract (AGE) is proven to have anticancer, antihepatotoxic, neuroprotective and neurotrophic properties. -Cystathionase (CTH), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) are involved in H2S/sulfane sulfur endogenous formation from L-cysteine. The aim of the study was to determine the effect of SAC on MCF-7 cells survival and apoptosis, which is a widely known approach to reduce the number of cancer cells. An additional goal of this paper was to investigate the effect of SAC on the activity and expression of enzymes involved in H2S production. The experiments were carried out in the human breast adenocarcinoma cell line MCF-7. Changes in the cell viability were determined by MTT assay. Cell survival was determined by flow cytometry (FC). Changes in enzymes expression were analyzed using Western blot. After 24 h and 48 h incubation with 2245 µM SAC, induction of late apoptosis was observed. A decrease in cell viability was observed with increasing SAC concentration and incubation time. SAC had no significant cytotoxic effect on the MCF-7 cells upon all analyzed concentrations. CTH, MPST and CBS expression were confirmed in non-treated MCF-7 cells. Significant decrease in MPST activity at 2245 µM SAC after 24 h and 48 h incubation vs. 1000 µM SAC was associated with decrease in sulfane sulfur levels. The presented results show promising SAC effects regarding the deterioration of the MCF-7 cells' condition in reducing their viability through the downregulation of MPST expression and sulfate sulfur level reduction.

Expression and activity of hydrogen sulfide generating enzymes in murine macrophages stimulated with lipopolysaccharide and interferon-γ.

Murine macrophages of the J774A.1 line are hydrogen sulphide-producing cells with the primary role of γ-cystathionase (CTH) and secondary role of 3-mercaptopyruvate sulfurtransferase (limited by cysteine availability) and with a negligible role of cystathionine ß-synthase (CBS) in H2S generation. J774A.1 cells stimulation with lipopolysaccharide (LPS) or interferon-gamma (IFNγ) resulted in decreased H2S levels after 24 h of incubation; however, they were restored to the control level after 48 h. Negligible CBS expression and activity in J774A.1 cells can result in homocysteine availability for CTH-catalyzed, H2S-generating reactions. This was supported by an increased CTH expression (IFNγ, 24 h and 48 h, and LPS, 48 h) and activity (24 h, LPS) in the stimulated cells. The results confirm the suggested feedback regulation between CBS and CTH.

Effect of Physical Exercise on the Febrigenic Signaling is Modulated by Preoptic Hydrogen Sulfide Production.

We tested the hypothesis that the neuromodulator hydrogen sulfide (H2S) in the preoptic area (POA) of the hypothalamus modulates the febrigenic signaling differently in sedentary and trained rats. Besides H2S production rate and protein expressions of H2S-related synthases cystathionine ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MPST) and cystathionine γ-lyase (CSE) in the POA, we also measured deep body temperature (Tb), circulating plasma levels of cytokines and corticosterone in an animal model of systemic inflammation. Rats run on a treadmill before receiving an intraperitoneal injection of lipopolysaccharide (LPS, 100 µg/kg) or saline. The magnitude of changes of Tb during the LPS-induced fever was found to be similar between sedentary and trained rats. In sedentary rats, H2S production was not affected by LPS. Conversely, in trained rats LPS caused a sharp increase in H2S production rate that was accompanied by an increased CBS expression profile, whereas 3-MPST and CSE expressions were kept relatively constant. Sedentary rats showed a significant LPS-induced release of cytokines (IL-1ß, IL-6, and TNF-α) which was virtually abolished in the trained animals. Correlation between POA H2S and IL-6 as well as TNF-α was observed. Corticosterone levels were augmented after LPS injection in both groups. We found correlations between H2S and corticosterone, and corticosterone and IL-1ß. These data are consistent with the notion that the responses to systemic inflammation are tightly regulated through adjustments in POA H2S production which may play an anti-inflammatory role downmodulating plasma cytokines levels and upregulating corticosterone release.

Upregulation of 3-MST Relates to Neuronal Autophagy After Traumatic Brain Injury in Mice.

3-mercaptopyruvate sulfurtransferase (3-MST) was a novel hydrogen sulfide (H2S)-synthesizing enzyme that may be involved in cyanide degradation and in thiosulfate biosynthesis. Over recent years, considerable attention has been focused on the biochemistry and molecular biology of H2S-synthesizing enzyme. In contrast, there have been few concerted attempts to investigate the changes in the expression of the H2S-synthesizing enzymes with disease states. To investigate the changes of 3-MST after traumatic brain injury (TBI) and its possible role, mice TBI model was established by controlled cortical impact system, and the expression and cellular localization of 3-MST after TBI was investigated in the present study. Western blot analysis revealed that 3-MST was present in normal mice brain cortex. It gradually increased, reached a peak on the first day after TBI, and then reached a valley on the third day. Importantly, 3-MST was colocalized with neuron. In addition, Western blot detection showed that the first day post injury was also the autophagic peak indicated by the elevated expression of LC3. Importantly, immunohistochemistry analysis revealed that injury-induced expression of 3-MST was partly colabeled by LC3. However, there was no colocalization of 3-MST with propidium iodide (cell death marker) and LC3 positive cells were partly colocalized with propidium iodide. These data suggested that 3-MST was mainly located in living neurons and may be implicated in the autophagy of neuron and involved in the pathophysiology of brain after TBI.

Expression of 3-Mercaptopyruvate Sulfurtransferase in the Mouse.

3-Mercaptopyruvate sulfurtransferase (MST) is one of the principal enzymes for the production of hydrogen sulfide and polysulfides in mammalians, and emerging evidence supports the physiological significance of MST. As a fundamental study of the physiology and pathobiology of MST, it is necessary to establish the tissue distribution of MST in mice. In the present study, the expression of MST in various organs of adult and fetal mice was analyzed by Western blotting and enzyme-immunohistochemistry. Moreover, the histology of MST gene-deficient mice was examined. Western blotting revealed that all organs examined had MST. The brain, liver, kidneys testes, and endocrine organs contained large amounts of MST, but the lungs, spleen, thymus, and small intestine did not. Immunohistochemically, the MST expression pattern varies in a cell-specific manner. In the brain, neural and glial cells are positively stained; in the lung, bronchiolar cells are preferentially stained; in the liver, hepatocytes around central veins are more strongly stained; renal convoluted cells are strongly stained; and pancreatic islets are strongly stained. Fetal tissues were studied, and MST expression was found to be similar before and after birth. Histological observation revealed no remarkable findings in MST gene-deficient mice. The present study revealed fundamental information regarding the MST expression of various organs in adult and fetal mice, and the morphological phenotype of MST gene-deficient mice.

Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 µM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 µM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 µM, IC50 = 267.4 µM; Ki = 0.84 µM, IC50 = 9.28 µM; and Ki = 0.592 µM, IC50 = 6.54 µM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 µg/ml. This information should be useful for the discovery of inhibitors of MtbBS.

Expression profile of hydrogen sulfide and its synthases correlates with tumor stage and grade in urothelial cell carcinoma of bladder.

BACKGROUND: Hydrogen sulfide (H2S) is a newly discovered gas transmitter. It is synthesized by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MPST). Endogenous hydrogen sulfide has never been studied in bladder cancer. PURPOSE: We evaluated H2S production and its synthases expression levels in transitional cell carcinoma (urothelial cell carcinoma of bladder [UCB]) of human bladder tissue and cell lines. MATERIALS AND METHODS: Immunostaining was performed in urothelial cell lines and bladder specimens from 94 patients with UCB of different stages/grades. The expression levels/activities of CBS, CSE, and MPST of specimens and cell lines were analyzed by image semiquantity assay, western blot, and a sulfur-sensitive electrode. We tried to find the correlation between hydrogen sulfide and its synthases with tumor stage in UCB. All experiments were repeated at least 3 times. RESULTS: Immunoreactivity for CBS, CSE, and MPST was detected in malignant uroepithelium and muscular layer of all tissues examined and cultured cells. The expression levels of CBS, CSE, and MPST were associated with UCB stage/grade. Muscle-invasive bladder cancer samples showed the highest production of H2S (52.6±2.91 nmol/[mg·min]) among all tested samples and EJ cells (transitional cell carcinoma, grade IIIshowed the highest production of H2S among all tested cell lines (53.3±7.02nmol/[mg·min]). CONCLUSIONS: Protein levels and catalytic activities of CBS, CSE, and MPST increased with the increase of malignant degrees in human bladder tissues and human UCB cell lines. Our findings may promote the application of these novel enzymes to UCB diagnosis or treatment.

Chronic intermittent hypoxia promotes expression of 3-mercaptopyruvate sulfurtransferase in adult rat medulla oblongata.

The present experiments were carried out to investigate the expression of 3-mercaptopyruvate sulfurtransferase (3MST) in medulla oblongata of rats and effects of chronic intermittent hypoxia (CIH) on its expression. Sprague Dawley adult rats were randomly divided into two groups, including control (Con) group and CIH group. The endogenous production of hydrogen sulfide (H2S) in medulla oblongata tissue homogenates was measured using the methylene blue assay method, 3MST mRNA and protein expression were analyzed by RT-PCR and Western blotting, respectively, and the expression of 3MST in the neurons of respiratory-related nuclei in medulla oblongata of rats was investigated with immunohistochemical technique. CIH elevated the endogenous H2S production in rat medulla oblongata (P<0.01). The RT-PCR and Western blotting analyses showed that 3MST mRNA and protein were expressed in the medulla oblongata of rats and CIH promoted their expression (P<0.01). Immunohistochemical staining indicated that 3MST existed in the neurons of pre-Bötzinger complex (pre-BötC), hypoglossal nucleus (12N), ambiguous nucleus (Amb), facial nucleus (FN) and nucleus tractus solitarius (NTS) in the animals and the mean optical densities of 3MST-positive neurons in the pre-BötC, 12N and Amb, but not in FN and NTS, were significantly increased in CIH group (P<0.05). In conclusion, 3MST exists in the neurons of medullary respiratory nuclei and its expression can be up-regulated by CIH in adult rat, suggesting that 3MST-H2S pathway may be involved in regulation of respiration and protection on medullary respiratory centers from injury induced by CIH.

Further evidence of endogenous hydrogen sulphide as a mediator of relaxation in human and rat bladder.

We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human and Sprague-Dawley (SD) rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine ß-synthase (CBS), cystathionine γ lyase (CSE) and 3-mercaptopyruvate sulphur transferase (MPST, as known as 3-MST) by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine (L-Cys, a substrate of CBS, CSE and MPST) elicited relaxation in a dose-dependent manner on human bladder strips pre-contracted by acetylcholine chloride. This effect could be diminished by the ATP-sensitive potassium ion (KATP) channel blocker glibenclamide (GLB), the CSE inhibitor DL-propargylglycine (PPG) and the CBS inhibitor hydroxylamine (HA). H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms (LUTS) or overactive bladder (OAB).

Brain 3-Mercaptopyruvate Sulfurtransferase (3MST): Cellular Localization and Downregulation after Acute Stroke.

3-Mercaptopyruvate sulfurtransferase (3MST) is an important enzyme for the synthesis of hydrogen sulfide (H2S) in the brain. We present here data that indicate an exclusively localization of 3MST in astrocytes. Regional distribution of 3MST activities is even and unremarkable. Following permanent middle cerebral artery occlusion (pMCAO), 3MST was down-regulated in both the cortex and striatum, but not in the corpus collosum. It appears that the down-regulation of astrocytic 3MST persisted in the presence of astrocytic proliferation due to gliosis. Our observations indicate that 3MST is probably not responsible for the increased production of H2S following pMCAO. Therefore, cystathionine ß-synthase (CBS), the alternative H2S producing enzyme in the CNS, remains as a more likely potential therapeutic target than 3MST in the treatment of acute stroke through inhibition of H2S production.

Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, apolipoprotein E-deficient mice.

OBJECTIVES: To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development. METHODS AND RESULTS: Heterozygous knockout mice for the lipoid acid synthase gene (Lias(+/-)) were crossed with apolipoprotein E-deficient (ApoE(-/-)) mice, and the plaque size in aortic sinuses of Lias(+/-)ApoE(-/-)mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias(+/-)ApoE(-/-) males were significantly larger (1.5x) than those in Lias(+/+) ApoE(-/-) littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/oxidized glutathione (GSH/GSSH) ratio, a systemic index of body redox balance. Lias(+/-)ApoE(-/-)males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias(+/-)ApoE(-/-)males. Female Lias(+/-)ApoE(-/-)also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females. CONCLUSIONS: Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduced antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.

The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.

Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis.

3-Mercaptopyruvate sulfurtransferase (MST) (EC 2.8.1.2), a multifunctional enzyme, catalyzes a transsulfuration from mercaptopyruvate to pyruvate in the degradation process of cysteine. A stoichiometric concentration of hydrogen peroxide and of tetrathionate (S(4)O(6)(2-)) inhibited rat MST (k(i) = 3.3 min(-1), K(i) = 120.5 microM and k(i) = 2.5 min(-1), K(i) = 178.6 microM, respectively). The activity was completely restored by dithiothreitol or thioredoxin with a reducing system containing thioredoxin reductase and NADPH, but glutathione did not restore the activity. On the other hand, an excess molar ratio dose of hydrogen peroxide inactivated MST. Oxidation with a stoichiometric concentration of hydrogen peroxide protected the enzyme against reaction by iodoacetate, which modifies a catalytic Cys(247), suggesting that Cys(247) is a target of the oxidants. A matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis revealed that hydrogen peroxide- and tetrathionate-inhibited MSTs were increased in molecular mass consistent with the addition of atomic oxygen and with a thiosulfate (S(2)O(3)(-)), respectively. Treatment with dithiothreitol restored modified MST to the original mass. These findings suggested that there was no nearby cysteine with which to form a disulfide, and mild oxidation of MST resulted in formation of a sulfenate (SO(-)) at Cys(247), which exhibited exceptional stability and a lower redox potential than that of glutathione. Oxidative stress decreases MST activity so as to increase the amount of cysteine, a precursor of thioredoxin or glutathione, and furthermore, these cellular reductants restore the activity. Thus the redox state regulates MST activity at the enzymatic level, and on the other hand, MST controls redox to maintain cellular redox homeostasis.

Molybdenum cofactor biosynthesis in humans: identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry.

The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N-terminus of the His6-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity.

Biotin synthase is catalytic in vivo, but catalysis engenders destruction of the protein.

Biotin synthase is responsible for the synthesis of biotin from dethiobiotin and sulfur. Although the name of the protein implies that it functions as an enzyme, it has been consistently reported that biotin synthase produces <1 molecule of biotin per molecule of protein in vitro. Moreover, the source of the biotin sulfur atom has been reported to be the [2Fe-2S] center of the protein. Biotin synthase has therefore been designated as a substrate or reactant rather than an enzyme. We report in vivo experiments demonstrating that biotin synthase is catalytic but that catalysis puts the protein at risk of proteolytic destruction.

Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study.

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe Mössbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.

Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use.

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5'-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.