Biosynthetic preparation of labeled 4,4-dimethylzymosterol.

Labeled 4,4-dimethyl-5 alpha -cholesta-8,24-dien-3 beta-ol (4,4-dimethylzymosterol) was prepared by incubating labeled mevalonate with rat liver extracts in the presence of arsenite and lanosterol.

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation.

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J.

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate.

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450.

Altered cytochrome P-450 in a yeast mutant blocked in demethylating C-32 of lanosterol.

Spectroscopic and enzymatic analysis of a Saccharomyces cerevisiae mutant in sterol biosynthesis (SG1 (erg 11); Trocha, P. J., Jasne, S.

The relation of heme to catalase apoprotein synthesis in yeast.

The synthesis of two hemoproteins, catalase A and catalase T, was studied in mutants of Saccharomyces cerevisiae deficient in heme formation. These mutants can be grown on the end-product heme or on a heme precursor, or on ergosterol and Tween 80 (a source of oleic acid). It was found by immunoprecipitation that, in the presence of heme, catalases A and T were present in the mutants, but that in its absence (growth on ergosterol and Tween 80) the apoproteins of these enzymes were not detectable.

Synthesis of the apoprotein of cytochrome b in heme-deficient yeast cells.

The presence of the apoprotein of cytochrome b has been demonstrated in a mutant of Sacchromyces cerevisiae lacking delta-aminolevulinic acid synthase and, hence, devoid of heme. The apoprotein of cytochrome b present in the mutant was identical with cytochrome b of control cells (mutant cells grown in the presence of delta-aminolevulinic acid) by the following criteria: similar apparent molecular weights in dodecyl sulfate-polyacrylamide gel electrophoresis; anomalous migration behavior during electrophoresis in polyacrylamide gels of different porosities; identical gel pattern obtained after immunoprecipitation with specific antiserum against cytochrome b; and identical fingerprints obtained after limited proteolysis with Staphylococcus aureus V8 protease. The kinetics of incorporation in vivo of [35S]methionine into apoprotein of cytochrome b in the mutant suggested that heme deficiency may affect assembly into the membrane of subunits of the cytochrome b.

5-Dehydro-3-deoxy-D-arabino-heptulosonic acid 7-phosphate. An intermediate in the 3-dehydroquinate synthase reaction.

3-Dehydroquinate synthase was purified to homogeneity from Escherichia coli. It was found to be a single polypeptide chain of Mr = approximately 57,000. Reaction mixtures of pure enzyme and the substrate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, were incubated for short times and treated with NaB3H4.

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed.

Yeast mutants blocked in removing the methyl group of lanosterol at C-14. Separation of sterols by high-pressure liquid chromatography.

Sterols of a nystatin resistant mutant of the wild type parent of Saccharomyces cerevisiae were separated by a newly developed procedure involving high-pressure liquid chromatography and were identified. The mutant contained larger amounts of squalene and lanosterol (I) than the wild type, as well as 4,14-dimethylcholesta-8,24-dien-3beta-ol (II), 4,14-dimethylergosta-8,24(28)-dien-3beta-ol (III), and 14-methylergosta-8,24(28)-dien-3beta-ol (IV), which were not hitherto found in yeast. These results indicated a block in removal of the methyl group at C-14 of lanosterol.

Stereochemical studies of the pyruvate kinase reaction with (Z)- and (E)-phosphoenol-alpha-ketobutyrate.

(Z)-and (E)-phosphoenol-2-ketobutyrate were synthesized. [3-2H]-2-Ketobutyrates were formed from both isomers in the pyruvate kinase reaction in 2H2O and were converted to chiral propionates. Authentic (2S)-[2-2H]propionic acid was also prepared, and the optical rotatory dispersion curves of the propionates were compared.

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme.

Properties of tyrosine-inhibitable 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthase from Salmonella.

Tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase was purified to homogeneity without significant loss of sensitivity to inhibition by tyrosine from an operator-constitutive strain (tyrOc) of Salmonella. The enzyme had an apparent molecular weight of 76,000 by gel filtration and a subunit molecular weight of 40,000 by sodium dodecyl sulfate-gel electrophoresis and by reaction with dimethyl suberimidate. It had an isoelectric point of 4.

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc).

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE.

tyrR, a regulatory gene of tyrosine biosynthesis in Salmonella typhimurium.

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which tyrosine pathway enzymes were not repressed by l-tyrosine. The mutants produced elevated levels of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase, and these enzymes as well as transaminase A were not repressed by high concentrations of tyrosine. Genetic analysis revealed that a mutation in a gene designated tyrR was responsible for the constitutivity of the tyrosine pathway enzymes in strains SG1, SG7, and SG9, and that tyrR was linked to pyrF.

Regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium.

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase (specified by pheA) was highly elevated. Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA, and conjugation and merodiploid analysis indicated that it was in the 95- to 100-min region of the Salmonella chromosome. Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR, a gene specifying a cytoplasmic product that affected pheA.

Enzyme alterations in tyrosine and phenylalanine auxotrophs of Salmonella typhimurium.

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase.

Mechanism and stereochemistry of 5-dehydroquinate synthetase.

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) labeled at C-7 randomly or stereospecifically with tritium and at C-1 with (14)C was converted enzymically to 5-dehydroquinate. Tritium of all three substrates was completely retained in 5-dehydroquinate, in accord with formation of a non-ketonizing 6,7-enol intermediate. The 5-dehydroquinates were dehydrated to 5-dehydroshikimate by 5-dehydroquinate dehydratase, which is known to catalyze a cis-elimination.