Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

BACKGROUND: In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS: PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS: It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS: Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems.

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form.

Human Mesenchymal Stem Cell Hydrogen Sulfide Production Critically Impacts the Release of Other Paracrine Mediators After Injury.

BACKGROUND: The use of mesenchymal stem cells (MSCs) for treatment during ischemia is novel. Hydrogen sulfide (H2S) is an important paracrine mediator that is released from MSCs to facilitate angiogenesis and vasodilation. Three enzymes, cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE), and 3-mercaptopyruvate-sulfurtransferase (MPST), are mainly responsible for H2S production. However, it is unclear how these enzymes impact the production of other critical growth factors and chemokines. We hypothesized that the enzymes responsible for H2S production in human MSCs would also critically regulate other growth factors and chemokines. MATERIALS AND METHODS: Human MSCs were transfected with CBS, MPST, CSE, or negative control small interfering RNA. Knockdown of enzymes was confirmed by polymerase chain reaction. Cells were plated in 12-well plates at 100,000 cells per well and stimulated with tumor necrosis factor-α (TNF-α; 50 ng/mL), lipopolysaccharide (LPS; 200 ng/mL), or 5% hypoxia for 24 h. Supernatants were collected, and cytokines measured by multiplex beaded assay. Data were compared with the Mann-Whitney U-test, and P < 0.05 was significant. RESULTS: TNF-α, LPS, and hypoxia effectively stimulated MSCs. Granulocyte colony-stimulating factor (GCSF), epidermal growth factor, fibroblast growth factor, granulocyte/monocyte colony-stimulating factor (GMCSF), vascular endothelial growth factor, and interferon gamma-inducible protein 10 were all significantly elevated when CSE was knocked down during TNF-α stimulation (P < 0.05). Knockdown of MPST during LPS stimulation more readily increased GCSF and epidermal growth factor but decreased GMCSF (P < 0.05). CBS knockdown decreased production of GCSF, fibroblast growth factor, GMCSF, and vascular endothelial growth factor (P < 0.05) after hypoxia. CONCLUSIONS: The enzymes that produce H2S in MSCs are also responsible for the production of other stem cell paracrine mediators under stressful stimuli. Therefore, reprogramming MSCs to endogenously produce more H2S as a therapeutic intervention could also critically impact other paracrine mediators, which may alter the desired beneficial effects.

Overexpression of endogenous lipoic acid synthase attenuates pulmonary fibrosis induced by crystalline silica in mice.

Oxidative stress and inflammatory processes are proposed to mediate the development of silicosis. However, antioxidant therapy has not produced consistent results during the treatment of silicosis. α-Lipoic acid synthesized by lipoic acid synthase is a powerful anti-oxidant and helps protect mitochondria. Thus far, the effect of endogenous α-Lipoic acid on silicosis has not been elucidated yet. We established an experimental model of silicosis with wildtype and LiasH/H mice, a new antioxidant mouse model which has overexpressed Lias gene (∼150 %) relative to its wild type counterpart. We systemically examined main pathological changes of pulmonary fibrosis, and explored α-lipoic acid effects on oxidative stress, inflammatory and pulmonary fibrosis biomarkers in silica-instillated mice. In LiasH/H mice over-expression of lipoic acid alleviated the severity of major pathological alterations in the early stage of pulmonary fibrosis induced by silica compared with wild type mice. Silica significantly increased oxidative stress in both wild type and LiasH/H mice. The antioxidant defense was strengthen including increased NRF2 and LIAS production in LiasH/H mice. Relieved oxidative stress resulted in decreased inflammatory response and secretion of chemokines. LiasH/H mice reduced chronic inflammatory response and inhibition of NF-κB activity after silica instillation. The LiasH/H mouse model overexpression of lipoic acid synthase gene retarded the development of silica-induced pulmonary fibrosis. Strengthen antioxidant defense by increased lipoic acid synthase is a potential strategy for protection against silica-induced pulmonary fibrosis.

Escherichia coli tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA via S-geranylated-intermediate.

To date the only tRNAs containing nucleosides modified with a selenium (5-carboxymethylaminomethyl-2-selenouridine and 5-methylaminomethyl-2-selenouridine) have been found in bacteria. By using tRNA anticodon-stem-loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA in a two-step process involving S2U-RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U-RNA (with SePO33- ). No 'direct' S2U-RNA→Se2U-RNA replacement is observed in the presence of SelU/SePO33- only (without ppGe). These results suggest that the in vivo S2U→Se2U and S2U→geS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events - geranylation (S2U→geS2U) and selenation (geS2U→Se2U).

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis.

Recent studies of hydrogen sulfide (H2S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme FeII persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

Fatty acids promote fatty liver disease via the dysregulation of 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide pathway.

OBJECTIVE: Accumulation of free fatty acids (FFAs) in hepatocytes induces lipotoxicity, leading to non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate the underlying mechanisms by which FFA contributes to the pathogenesis of NAFLD via the regulation of 3-mercaptopyruvate sulfurtransferase (MPST), a key enzyme that regulates endogenous hydrogen sulfide (H2S) biosynthesis. DESIGN: Hepatic MPST expression was evaluated in mice and patients with NAFLD. A variety of molecular approaches were used to study the effects of MPST regulation on hepatic steatosis in vivo and in vitro. RESULTS: In vitro treatment of hepatocytes with FFAs upregulated MPST expression, which was partially dependent on NF-κB/p65. Hepatic MPST expression was markedly increased in high fat diet (HFD)-fed mice and patients with NAFLD. Partial knockdown of MPST via adenovirus delivery of MPST short hairpin RNA or heterozygous deletion of the Mpst gene significantly ameliorated hepatic steatosis in HFD-fed mice. Consistently, inhibition of MPST also reduced FFA-induced fat accumulation in L02 cells. Intriguingly, inhibition of MPST significantly enhanced rather than decreased H2S production, whereas MPST overexpression markedly inhibited H2S production. Co-immunoprecipitation experiments showed that MPST directly interacted with and negatively regulated cystathionine γ-lyase (CSE), a major source of H2S production in the liver. Mechanistically, MPST promoted steatosis via inhibition of CSE/H2S and subsequent upregulation of the sterol regulatory element-binding protein 1c pathway, C-Jun N-terminal kinase phosphorylation and hepatic oxidative stress. CONCLUSIONS: FFAs upregulate hepatic expression of MPST and subsequently inhibit the CSE/H2S pathway, leading to NAFLD. MPST may be a potential therapeutic target for NAFLD.

Multiple role of 3-mercaptopyruvate sulfurtransferase: antioxidative function, H2 S and polysulfide production and possible SOx production.

Rat 3-mercaptopyruvate sulfurtransferase (MPST) is a 32 808 Da simple protein. Cys247 is a catalytic site, and Cys154 and Cys263 are on the enzyme surface. MPST is found in all tissues, particularly in the kidneys, although the localization of its activity differs in each tissue. In this review, four functions of MPST are reviewed: (i) antioxidative function: Cys247 is redox-sensitive and serves as a redox-sensing switch. It is oxidized to cysteine sulfenate, which has a low redox potential, upon which the enzyme is inactivated. Then, reduced thioredoxin (Trx) with a reducing system (Trx reductase and NADPH) reduces the sulfenate to restore activity; meanwhile, Cys154 and Cys263 form an intermolecular disulfide bond, which serves as another redox-sensing switch. Consequently, Trx specifically cleaves the intermolecular disulfide bond by converting it from the inactive form (dimer) to the active form (monomer). (ii) Hydrogen sulfide and polysulfide production: hydrogen sulfide is produced via reduction of the persulfurated sulfur-acceptor substrate by reduced Trx or Trx with a reducing system; as an alternative process, stable polysulfurated or persulfurated Cys247 as a reaction intermediate is reduced by Trx with a reducing system to release hydrogen sulfide and polysulfides. (iii) Possible sulfur oxide production: sulfur oxides (SO, SO2 and SO3 ) can be produced in the redox cycle of sulfane sulfur formed at the catalytic site Cys247 (Cys-SO- , Cys-SO2- and Cys-SO3- ) as reaction intermediates and released by reduced Trx or Trx with a reducing system. (iv) Possible anxiolytic-like effects: MPST-knockout mice exhibited anxiolytic-like effects.

Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli.

Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders ∆fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in ∆fur or ∆sodA ∆sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.

H2S Synthesizing Enzymes: Biochemistry and Molecular Aspects.

Hydrogen sulfide (H2S) is a biologically active gas that is synthesized naturally by three enzymes, cystathionine γ-lyase (CSE), cystathionine ß-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). These enzymes are constitutively present in a wide array of biological cells and tissues and their expression can be induced by a number of disease states. It is becoming increasingly clear that H2S is an important mediator of a wide range of cell functions in health and in disease. This review therefore provides an overview of the biochemical and molecular regulation of H2S synthesizing enzymes both in physiological conditions and their modulation in disease states with particular focus on their regulation in asthma, atherosclerosis and diabetes. The importance of small molecule inhibitors in the study of molecular pathways, the current use of common H2S synthesizing enzyme inhibitors and the relevant characteristics of mice in which these enzymes have been genetically deleted will also be summarized. With a greater understanding of the molecular regulation of these enzymes in disease states, as well as the availability of novel small molecules with high specificity targeted towards H2S producing enzymes, the potential to regulate the biological functions of this intriguing gas H2S for therapeutic effect can perhaps be brought one step closer.

Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons.

Hydrogen sulfide (H2 S) is a gaseous neuromodulator produced from L-cysteine. H2 S is generated by three distinct enzymatic pathways mediated by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H2 S production in PC12 cells (rat pheochromocytoma-derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H2 S were produced from L-cysteine in the presence of α-ketoglutarate, together with dithiothreitol. The production of H2 S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L-aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H2 S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H2 S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H2 S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H2 S generation. In the peripheral nervous system, hydrogen sulfide (H2 S) has been implicated in neurogenic pain or hyperalgesia. This study provides evidence that H2 S is synthesized in peripheral neurons through two mitochondrial enzymes, cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MPST). We propose that mitochondrial metabolism plays key roles in the physiology and pathophysiology of the peripheral nervous system via regulation of neuronal H2 S production.

A metazoan ATAC acetyltransferase subunit that regulates mitogen-activated protein kinase signaling is related to an ancient molybdopterin synthase component.

Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex.

The structure and NO binding properties of the nitrophorin-like heme-binding protein from Arabidopsis thaliana gene locus At1g79260.1.

The protein from Arabidopsis thaliana gene locus At1g79260.1 is comprised of 166-residues and is of previously unknown function. Initial structural studies by the Center for Eukaryotic Structural Genomics (CESG) suggested that this protein might bind heme, and consequently, the crystal structures of apo and heme-bound forms were solved to near atomic resolution of 1.32 A and 1.36 A, respectively. The rate of hemin loss from the protein was measured to be 3.6 x 10(-5) s(-1), demonstrating that it binds heme specifically and with high affinity. The protein forms a compact 10-stranded beta-barrel that is structurally similar to the lipocalins and fatty acid binding proteins (FABPs). One group of lipocalins, the nitrophorins (NP), are heme proteins involved in nitric oxide (NO) transport and show both sequence and structural similarity to the protein from At1g79260.1 and two human homologues, all of which contain a proximal histidine capable of coordinating a heme iron. Rapid-mixing and laser photolysis techniques were used to determine the rate constants for carbon monoxide (CO) binding to the ferrous form of the protein (k'(CO) = 0.23 microM(-1) s(-1), k(CO) = 0.050 s(-1)) and NO binding to the ferric form (k'(NO) = 1.2 microM(-1) s(-1), k(NO) = 73 s(-1)). Based on both structural and functional similarity to the nitrophorins, we have named the protein nitrobindin and hypothesized that it plays a role in NO transport. However, one of the two human homologs of nitrobindin contains a THAP domain, implying a possible role in apoptosis. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Mice with heterozygous deficiency of lipoic acid synthase have an increased sensitivity to lipopolysaccharide-induced tissue injury.

Alpha-lipoic acid (1, 2-dithiolane-3-pentanoic acid; LA), synthesized in mitochondria by LA synthase (Lias), is a potent antioxidant and a cofactor for metabolic enzyme complexes. In this study, we examined the effect of genetic reduction of LA synthesis on its antioxidant and anti-inflammatory properties using a model of LPS-induced inflammation in Lias+/- mice. The increase of plasma proinflammatory cytokine, TNF-alpha, and NF-kappaB at an early phase following LPS injection was greater in Lias+/- mice compared with Lias+/+ mice. The circulating blood white blood cell (WBC) and platelet counts dropped continuously during the initial 4 h. The counts subsequently recovered partially in Lias+/+ mice, but the recovery was impaired totally in Lias+/- mice. Administration of exogenous LA normalized the recovery of WBC counts in Lias+/- mice but not platelets. Enhanced neutrophil sequestration in the livers of Lias+/- mice was associated with increased hepatocyte injury and increased gene expression of growth-related oncogene, E-selectin, and VCAM-1 in the liver and/or lung. Lias gene expression in tissues was 50% of normal expression in Lias+/- mice and reduced further by LPS treatment. Decreased Lias expression was associated with diminished hepatic LA and tissue oxidative stress. Finally, Lias+/- mice displayed enhanced mortality when exposed to LPS-induced sepsis. These data demonstrate the importance of endogenously produced LA for preventing leukocyte accumulation and tissue injury that result from LPS-induced inflammation.

Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity.

The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI. We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E. coli 83-1 cells. The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E. coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes. By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E. coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells. The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH.

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

Coupling cell growth, proliferation, and death. Hippo weighs in.

Four recent papers describe the characterization in Drosophila of Hippo, a serine/threonine kinase of the Sterile 20 (STE20) group, resembling Mst1 and Mst2. Hippo restricts cell growth and cell proliferation, promotes cell death, and interacts with the tumor suppressors Salvador and Warts. This, together with the ability of Mst2 to rescue hippo mutant phenotypes, argues that Mst/Hippo proteins are tumor suppressors.

Escherichia coli MoeA and MogA. Function in metal incorporation step of molybdenum cofactor biosynthesis.

Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor. Here we show that moeA(-) and mogA(-) cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide. Human sulfite oxidase expressed in E. coli moeA(-) could be activated in vitro in the presence of MoeA and low concentrations of molybdate. Sulfite oxidase purified from the moeA(-) lysate was also activated, although to a lesser extent than observed in the presence of lysate. MogA was incapable of activating sulfite oxidase expressed in E. coli mogA(-). These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation.

Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein.

The product of the miaB gene, MiaB, from Escherichia coli participates in the methylthiolation of the adenosine 37 residue during modification of tRNAs that read codons beginning with uridine. A His-tagged version of MiaB has been overproduced and purified to homogeneity. Gel electrophoresis and size exclusion chromatography revealed that MiaB protein is a monomer. As isolated MiaB contains both iron and sulfide and an apoprotein form can chelate as much as 2.5-3 iron and 3-3.5 sulfur atoms per polypeptide chain. UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions, whereas [2Fe-2S] and [3Fe-4S] forms are generated under aerobic conditions. Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. Together with the previously shown requirement of S-adenosylmethionine (AdoMet) for the methylthiolation reaction, the finding that MiaB is an iron-sulfur protein suggests that it belongs to a superfamily of enzymes that uses [Fe-S] centers and AdoMet to initiate radical catalysis. MiaB is the first and only tRNA modification enzyme known to contain an Fe-S cluster.