Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared repressed while sulphite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) were derepressed and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.Abbreviations APS adenylylphosphosulphate - MVH reduced methylviologen - OAS O-acetyl-l-serine - PAPS 3-phospho adenylylphosulphate     

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae

A number of strains of Saccharomyces which produce sulphite by sulphate reduction were examined from an enzymatic and genetic point of view.There are a number of mechanisms that regulate this activity. All of these mechanisms involve the sulphite-reducing activity. In the strains examined, reduced function as a result of mutation in the Sr-locus (affecting H₂S-NADP oxidoreductase EC 1.8.1.2), repression of biosynthesis of the enzyme because of a mutation below the specific locus, and inhibition of the enzyme by endogenous factors were found to be responsible. The production of sulphite can also be connected with a complex state of heterozygosity.It is probably this multiplicity of biochemical and genetic mechanisms that accounts for the frequency with which the production of sulphite is observed in wild strains in nature.This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

The influence of pH on sulphite formation by yeasts

The influence of the initial pH of the substrate on the sulphite formation of three low-sulphite-and five high-sulphite-forming yeasts is described. Four distinctly different groups become apparent. The need for better evaluation of pure culture wine yeasts is stressed.     

The transformation of inorganic sulfur compounds and the assimilation of organic and inorganic carbon by the sulfur disproportionating bacterium Desulfocapsa sulfoexigens

The physiology of the sulfur disproportionator Desulfocapsa sulfoexigens was investigated in batch cultures and in a pH-regulated continuously flushed fermentor system. It was shown that a sulphide scavanger in the form of ferric iron was not obligatory and that the control of pH allowed production of more biomass than was possible in carbonate buffered but unregulated batch cultures. Small amounts of sulphite were produced during disproportionation of elemental sulfur and thiosulphate. In addition, it was shown that in the presence of hydrogen, a respiratory type of process is favored before the disproportionation of sulphite, thiosulphate and elemental sulfur. Sulphate reduction was not observed. D. sulfoexigens assimilated inorganic carbon even in the presence of organic carbon sources. Inorganic carbon assimilation was probably catalyzed by the reverse CO-dehydrogenase pathway, which was supported by the constitutive expression of the gene encoding CO-dehydrogenase in cultures grown in the presence of acetate and by the high carbon fractionation values that are indicative of this pathway.     

Effect of sulphite on superoxide dismutase activity in the mycorrhizal fungus Rhizopogon roseolus

 Sulphite at a concentration of 1 mM did not strongly affect the growth of mycelium. Higher concentrations of 5–20 mM almost completely inhibited the growth of mycelium and superoxide dismutase (SOD) [EC.1.15.1.1.] activity. The activity of this enzyme was not detectable on polyacrylamide gels. The lack of induction of SOD and the resulting oxidative stress may in part be responsible for the growth inhibition caused by high concentrations of sulphite. Accepted: 20 July 1995     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

Yeast PAPS reductase: properties and requirements of the purified enzyme

The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (M_R 80–85 k) is represented by a dimer which reduces 3-phosphoadenylyl sulphate to adenosine-3,5-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for bound-sulphite(s) as intermediate. V _max of the enriched enzyme was 4–7 nmol sulphite · min^-1 · mg^-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite · min^-1 · mg^-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The K _m of homologous thioredoxin was 0.6 · 10^-6 M, for the heterologous cosubstrate it increased to 1.4 · 10^-6 M. The affinity for PAPS remained practically unaffected (K _{m PAPS}: 19 · 10^-6 M in the homologous, and 21 · 10^-6 M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.Abbreviations APS adenylyl sulphate - DTE dithioerythritol - DTT dithiothreitol - HPLC high performance liquid chromatography - IEF isoelectric focusing - LSC liquid scintillation counting - 3,5-PAP adenosine-3,5-bisphosphate - PAPS 3-phosphoadenylyl sulphate - PEP phospho-(enol)pyruvate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - Tris 2-amino-2-hydroxymethyl-1,3-propanediol