Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high K_m for O-acetylserine and low K_m for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.     

Cysteine <Emphasis Type="Italic">S</Emphasis>-conjugate β-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds,xenobiotics and anticancer agents

Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1–15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M _r cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated.     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.     

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Voltage-gated Na⁺ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na⁺ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na⁺ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.     

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a β-barrel containing 19 membrane spanning β-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential β-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 β-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Computational Analysis of Cysteine Substitutions Modelled on the α- and β-domains of Cd5,Zn2-Metallothionein 2

Several mutant forms of rat liver Cd₅,Zn₂-metallothionein 2 (Cd₅,Zn₂-MT 2) [1] have been computationally modelled and analysed. All terminal cysteines (5, 13, 19, 21, 26, 29, 33, 36, 41, 48, 57 and 59, Figure 1) have been independently substituted by three other co-ordinating amino-acids (aspartate, glutamate and histidine), and the side-chains of the mutated residues have been modelled to co-ordinate the seven metal ions while minimizing the conformational variations with respect to the wild type protein. We have compared the ability of the putative mutant forms to maintain the MT binding properties. Substitution by aspartate residue best preserves the 3D MT structure. In addition, the mutations C5H plus C21H/E/D show neighbouring impairments that prevent their simultaneous substitution. Although replacement of cysteine by aspartate is feasible in all cases, to our knowledge there is no example of aspartate and cysteine residues co-ordinating to the same zinc atom. Accordingly, the use of histidine or glutamate instead of aspartate cannot be ruled out. The mutant forms in the -domain of Cd₅,Zn₂-MT 2 have yielded more neighbouring contacts than those in the -domain, which is corroborated by the accessible surface areas [2] of the sulfur atoms [3] in the native form.Abbreviations MT metallothionein - CD₅,Zn₂-MT Cadmium, Zinc-metallothionein - RMSD Root Mean Square Deviation - PDB Protein Data Bank - FEP Free Energy Perturbation - CnX mutant form of cysteine n (n = residue number) substituted by X (X = H, E or D, with H = histidine, E = glutamate, D = aspartate) - CnX/Y mutant forms CnX and CnY     

Cysteine 38 holds the key to NF-κB activation

The importance of parallel signaling pathways controlling NF-κB subunit posttranslational modifications is demonstrated by Sen et al. (2012), who reveal that RelA (p65) sulfhydration, at its highly conserved cysteine 38 residue, regulates association with the coactivator RPS3, DNA binding, and antiapoptotic gene expression.     

Surface Exposed Free Cysteine Suppresses Crystallization of Human γD-Crystallin

Human γD-crystallin (HGD) has remarkable stability against condensation in the human lens, sometimes over a whole lifetime. The native protein has a surface exposed free cysteine that forms dimers (Benedek, 1997; Ramkumar et al., 1864)^1,2 without specific biological function and leads to further protein association and/or aggregation, which creates a paradox for understanding its stability. Previous work has demonstrated that chemical modification of the protein at the free cysteine (C110), increases the temperature at which liquid–liquid phase separation occurs (LLPS), lowers protein solubility and suggests an important role for this amino acid in maintaining its long-term resistance to condensation. Here we demonstrate that mutation of the cysteine does not alter the structure or solubility (liquidus) line for the protein, but dramatically increases the protein crystal nucleation rate following LLPS, suggesting that the free cysteine has a vital role in suppressing crystallization in the human lens.     

Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Down-regulation and Covalent Binding at Cysteine 303 of β-Tubulin

Withaferin A (WA), a C₅,C₆-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G₂ and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C₆,C₇-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys³⁰³ of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.     

Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.     

Novel Modification of 5-Formyluracil by Cysteine Derivatives in Aqueous Solution‡

Abstract

Reactivities of 5-formyl-2′-deoxyuridine (fdU) and its 5′-monophosphate (fdUMP) to amino acids, amines and thiol compounds in neutral aqueous solution have been studied to elucidate the postmodification of the 5-formyluracil (fU) moiety in cells. fdU and fdUMP specifically reacted with cysteine and its analogs to form thiazolidine derivatives. The reaction involved condensation of the formyl group of fU with both α-NH₂ (or NH₂ at the equevalent position) and SH groups of cysteine derivatives.

     

Quercetin Targets Cysteine String Protein (CSPα) and Impairs Synaptic Transmission

Background

Cysteine string protein (CSPα) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPα is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPα in mice results in knockout mice that are normal for the first 2–3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPα prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPα represents a promising therapeutic target for the prevention of neurodegenerative disorders.

Methodology/Principal Findings

Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPα-CSPα dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPα dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPα function. Quercetin''s action on CSPα is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPα:Hsc70 units (70kDa heat shock cognate protein).

Conclusions/Significance

Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPα and the undesired consequences of CSPα dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPα.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Cysteine biosynthesis pathway in the archaeon Methanosarcina barkeri encoded by acquired bacterial genes?

The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic Escherichia coli strain NK3 led to the isolation of the Methanosarcina barkeri cysK gene [encoding O-acetylserine (thiol)-lyase-A], which displays great similarity to bacterial cysK genes. Adjacent to cysK is an open reading frame orthologous to bacterial cysE (serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like cysM genes [encoding O-acetylserine (thiol)-lyase-B] in Pyrococcus spp., Sulfolobus solfataricus, and Thermoplasma acidophilum. However, no orthologs for these genes can be found in Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.     

Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases

p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H₂ ¹⁸O to liberate galactose containing ¹⁸O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Preparation of Aryl β-d-Mannopyranoside Derivatives via β-d-arabino-Hexopyranosiduloses

Sequential oxidation and reduction of aryl 4, 6-O-benzylidene-β-d-glucosides with dimethyl sulfoxide-phosphorus pentoxide mixture (DMSO–P₂O₅) and sodium borohydride were carried out as a new means for the preparation of aryl β-d-mannopyranoside derivatives. p-Nitrophenyl 4, 6-O-benzylidene-β-d-mannopyranoside was obtained in 22% yield from the corresponding glucoside 3-O-acetate, whereas from the unprotected acetal, 4, 6-O-benzylidene acetals of the corresponding mannoside and alloside were isolated in the yields of 6.7 and 2.1%, respectively. Similarly, phenyl 4, 6-O-benzylidene β-d-mannoside, alloside, and altroside were obtained from the corresponding glucoside in 2.2, 0.8 and 2.1% yields, respectively.