Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.     

Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B.

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.     

大肠杆菌glgC基因的定点突变及功能分析

摘要: 利用PCR从Escherichia coli JM109基因组中扩增到全长为1 296 bp的glgC基因编码区, 通过PCR重组方法进行点突变, 获得氨基酸突变的3个突变体, 分别是Pro295Ser(Val121Ala, Met151Ile和Val334Asp)、Gly336 Asp单点突变和Pro295Ser/Gly336Asp(Lys109Arg), 其基因分别命名为295⁺³、336和295/ 336⁺¹。将突变和未突变的基因分别克隆到pET32-a, 构建重组质粒pET-glgC、pET-295⁺³、pET-336和pET-295/ 336+1, 在文中分别简称为a、b、c和d。转化大肠杆菌BL21(DE3), 在1 mmol/L IPTG 诱导下表达。SDS- PAGE 电泳分析显示, 在约67 kDa 处有1条明显与预期大小一致的蛋白质, 表明目的基因已得到融合表达。上述转化子的碘染和糖原含量测定结果, 第336位的Gly变成Asp后, 宿主菌的糖原含量提高; Pro295Ser/Gly336Asp(Lys109Arg)的突变导致宿主菌的糖原含量与Gly336Asp突变体相近, 表明在336突变基因的基础上增加Pro295Ser的突变没有进一步加大宿主菌中AGPase酶的反馈抑制效应的降低。已有的结果显示, Pro295Ser可以降低AGPase酶的反馈抑制效应活性, 而实验中295⁺³突变基因转入宿主菌后细胞糖原含量明显降低, 推测这个结果可能是295⁺³中的Val334Asp的突变造成, 而334位的氨基酸可能是AGPase功能域中的一个重要位点。     

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 µM compared to 50% protection by 405 µM 3PGA. Partial modification of the enzyme using [¹⁴C]-phenylglyoxal leads to a decrease in bothV _max,A _0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [¹⁴C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.     

Escherichia coli E-39 ADPglucose synthetase has different activation kinetics from the wild-type allosteric enzyme

Kinetic and binding studies have shown that Lys39 of Escherichia coli ADPglucose synthetase is involved in binding of the allosteric activator. In order to study structure-function relationships at the activator binding site, this lysine residue was substituted by glutamic acid (Lys39----Glu) by site-directed mutagenesis. The resultant mutant enzyme (E-39) showed activation kinetics different from those of the wild-type enzyme. The level of activation of the E-39 enzyme by the major activators of E. coli ADPglucose synthetase, 2-phosphoglycerate, pyridoxal phosphate, and fructose-1,6-phosphatase was only approximately 2-fold compared to activation of 15- to 28-fold respectively, for the wild-type enzyme. NADPH, an activator of the wild-type enzyme, was unable to activate the mutant enzyme. In addition, the concentrations of the above activators necessary to obtain 50% of the maximal stimulation of enzyme activity (A0.5) were 5-, 9-, and 23-fold higher, respectively, than those for the wild-type enzyme. The E-39 enzyme also had a lower apparent affinity (S0.5) for the substrates ATP and MgCl2 than the wild-type enzyme and the values obtained in the presence or absence of activator were similar. The concentration of inhibitor giving 50% of enzyme activity (I0.5) was also similar for the E-39 enzyme in the presence or absence of activator. These results indicate that the E-39 mutant enzyme is not effectively activated by the major activators of the E. coli ADPglucose synthetase wild-type enzyme, and that this amino acid substitution also prevents the allosteric effect that the activator has on the wild-type enzyme kinetics, either increasing its apparent affinity for the substrates or modulating the enzyme's sensitivity to inhibition.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations. MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity. ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively. ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.