Biochemical-genetic study of the first enzyme of histidine biosynthesis in Salmonella typhimurium: substrate and feedback binding regions.

Twenty-five strains of Salmonella typhimurium containing different mutations in the first gene of histidine biosynthesis were studied to correlate regions of the genetic map with biochemical functions. These strains contained either missense, double-frameshift, or suppressed nonsense mutations, all of which resulted in altered, though active, enzymes. Each mutant enzyme was assayed for activity in the presence of varying concentrations of the feedback inhibitor L-histidine or the substrates ATP and 5-phosphoribosyl-1-pyrophosphate. The feedback properties and substrate kinetics of each mutant enzyme were compared to wild-type values, and these results indicated that the following functions were correlated with regions of the hisG gene: feedback inhibition in two general areas, including regions IA and IB and regions V, VI, and VII; ATP binding in two general areas, including regions IA, IB, and II and regions V, VI, and VII; and 5-phosphoribosyl-1-pyrophosphate binding in two general areas, including regions IB, II, and III and regions V and VI.     

Composition of the first enzyme of histidine biosynthesis isolated from wild-type and mutant operator strains of Salmonella typhimurium.

The first enzyme of histidine biosynthesis in Salmonella typhimurium, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), has been purified from two bacterial strains containing histidine operator deletions and compared to the eenzyme from a strain that has a normal operator. The enzymes isolated in different ways also are compared. Evidence as to the separateness of the operator and first structural gene or covalent modification of the first enzyme was sought. Specific activity, histidine feedback inhibition, amino acid analysis, discontinuous-gel electrophoresis, and gel filtration of the native enzyme, and Ouchterlony double-immunodiffusion tests were carried out. The purified enzyme contains little phosphorous and has five cysteine residues per subunit, which all are readily titratable. No evidence for differences in the enzyme preparations was obtained. Thus, no evidence for overlap of the histidine operator with the first structural gene was obtained.     

Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis

Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme.     

D-amino acid dehydrogenase: the enzyme of the first step of D-histidine and D-methionine racemization in Salmonella typhimurium

Summary Mutants ofSalmonella typhimurium deficient in D-amino acid dehydrogenase were isolated in histidine auxotrophs able to utilize D-histidine(his-dhuA) 1. The mutants have lost the ability to utilize D-histidine and D-methionine due to mutations in the locusdadA mapped in co-transducible vicinity of the genehemA. ThedadA mutants were unable to deaminate D-histidine, D-methionine, D-alanine and several other D-amino acids to the respective keto products. Indad + strains the enzyme activity was the highest in toluenized cells. In crude sonieates it was 5 to 10 times less. Reduction of artificial electron accepters in the presence of D-amino acids behaved similarly. Keto product formation was strongly inhi-bited by cyanide. It has been concluded thereof that the deaminating enzyme is a D-amino acid dehydrogenase, the activity of which depends on structural integrity of a cell component or on a structure-bound electron accepter. The enzyme activity was inducible by adding L-or D-alanine to growth media. The induction was the highest in media with poor carbon sources. A temperature-sensitivedadA mutant was isolated. I t mapped indadA and had thermolabile D-amino acid dehydrogenase. This has indicated thatdadA is structural gene for the D-amino acid dehydrogenase. This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1. The nomenclature rules for describing genotypes and phenotypes of Demerecet al. (1966) were followed throughout this paper. E.g.dhuA ⁻ hisP⁺ mutants have Dhu⁺ phenotype, those with dhuA^{s - hisP s - mutations are phenotypically Dhu}- All strains with wild-type dhuA⁺ lOCUS are Dhu⁻.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli.

The Escherichia coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the first committed step of peptidoglycan biosynthesis (J. L. Marquardt, D. A. Siegele, R. Kolter, and C. T. Walsh, J. Bacteriol. 174:5748-5752, 1992). The map position of murZ (69.3 min) differed from that determined for murA (90 min), a gene which had been previously proposed to encode the same activity (P.S. Venkateswaran and H. C. Wu, J. Bacteriol. 110:935-944, 1972). Here we describe the construction of a chromosomal deletion of murZ and a plasmid containing murZ under arabinose control. Growth of cells containing the murZ deletion was dependent on the expression of murZ from the plasmid. We conclude that murZ is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E. coli. To simplify the nomenclature, we recommend that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned.     

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.     

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.     

Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.

The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.     

Enhancing the first enzymatic step in the histidine biosynthesis pathway increases the free histidine pool and nickel tolerance in Arabidopsis thaliana

Naturally selected nickel (Ni) tolerance in Alyssum lesbiacum has been proposed to involve constitutively high levels of endogenous free histidine. Transgenic Arabidopsis thaliana expressing a Salmonella typhimurium ATP phosphoribosyl transferase enzyme (StHisG) resistant to feedback inhibition by histidine contained approximately 2-fold higher histidine concentrations than wild type plants. Under exposure to a toxic Ni concentration, biomass production in StHisG expressing lines was between 14- and 40-fold higher than in wild-type plants. This suggested that enhancing the first step in the histidine biosynthesis pathway is sufficient to increase the endogenous free histidine pool and Ni tolerance in A. thaliana.