D.B. Sprinson

A single amino acid substitution converts cytochrome P45014DM to an inactive form, cytochrome P450SG1: Complete primary structures deduced from cloned DNAs

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.

The Biosynthesis of Aromatic Compounds from D-Glucose

Publisher Summary This chapter discusses the several aspects of the formation of shikimate, and with its conversion to aromatic amino acids. Although the role of D-glucose is stressed in the title, it should be pointed out that pyruvate and acetate, the starting materials for the other known pathways, may also be derived ultimately from D-glucose. A bacterial culture in a minimal medium (salts plus D-glucose) was irradiated with ultraviolet light, and the resultant mixture of wild-type and mutant strains was grown on a medium supplemented with yeast extract and casein hydrolyzate. Mutant strains were obtained which required for growth phenylalanine, tyrosine, tryptophan, and p -aminobenzoic acid. For certain of these strains, it was found that, of more than 50 compounds tested, only shikimic acid was able to replace the required aromatic supplement; other strains accumulated shikimic acid in the culture medium. It was therefore concluded that the former can convert shikimic acid to the aromatic metabolites, the genetic block in these organisms being somewhere before shikimic acid; the latter clearly were blocked after shikimic acid. Certain multiple aromatic auxotrophs grew slowly on the quadruple aromatic supplement, but were stimulated by the further addition of a trace of shikimic acid or of wild-type filtrate. The compound responsible for this effect was shown to be p -hydroxybenzoic acid, a bacterial vitamin subsequently found to be involved in the biosynthesis of methionine and lysine.

Yeast mutants requiring ergosterol as only lipid supplement

Abstract Mutants of Saccharomyces cerevisiae were isolated which required ergosterol or cholesterol as the only lipid supplement. They also required methionine, were petite, and showed complete absence of respiratory cytochromes. Revertants of these strains grew without ergosterol and methionine, were grande, and had respiratory cytochromes. Most revertants did not make ergosterol and were nystatin resistant. Sterol analysis and enzyme assays suggested a block in sterol formation after lanosterol.

Novel sterols in ergosterol deficient yeast mutants

Summary Sterols of nystatin resistant and ergosterol requiring mutants, and of the wild type parent of Saccharomyces cerevisiae were separated by a newly developed procedure and were identified. The mutants contained larger amounts of lanosterol (I) than the wild type, as well as 4, 14-dimethylcholesta-8, 24-dien-3β-ol (II), 4, 14-dimethylergosta-8, 24(28)-dien-3β-ol (III), and 14-methylergosta-8, 24(28)-dien-3β-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.

The stereochemistry of pyruvate kinase

Abstract α-Ketobutyrate enol phosphate consisting of approximately 80% cis and 20% trans isomers was prepared by the Perkow reaction. It was converted by pyruvate kinase in 2H2O, to α-ketobutyrate which on oxidative decarboxylation with hydrogen peroxide gave largely (2 R )-2- 2 H-propionate containing one atom of deuterium. It was concluded that protonation in the pyruvate kinase reaction occurred on the si face with cis -α-ketobutyrate enol phosphate as substrate, and probably also with the trans isomer.

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. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.

Synthesis of 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate by phosphorylation of methyl (methyl 3-deoxy-d-arabino-heptulopyranosid)onate

Abstract 3-Deoxy- d - arabino -2-heptulosonic acid 7-phosphate ( 5 ), the first committed intermediate in aromatic amino acid biosynthesis, has been synthesized in good yield by treatment of methyl (methyl 3-deoxy- d - arabino -2-heptulopyranosid)onate with diphenylphosphoric chloride under mild conditions to give the 7-diphenyl phosphate. Catalytic removal of the phenyl residues, followed by base-catalyzed hydrolysis resulted in formation of (methyl 3-deoxy- d - arabino -2-heptulopyranosid)onic acid dihydrogen 7-phosphate ( 4 ), which yielded a crystalline tris-(cyclohexylammonium) salt. Acid-catalyzed hydrolysis of 4 afforded 5 , which was used to purify 3-dehydroquinate synthase.

A regulatory mutation in tyrosine biosynthesis

Abstract Mutants of Salmonella with an altered control of the tyrosine repressible enzymes were obtained by selection for resistance to 4-fluoro-phenylalanine. The tyrosine repressible 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase and prephenate dehydrogenase (specified by aroF , and tyr , respectively) were constitutively and co-ordinately derepressed. The mutation to resistance was co-transducible with aroF and tyr . A unit of regulation in tyrosine biosynthesis in indicated.

The conversion of ethanolamine to acetate in mammalian tissues.

Abstract Ethanolamine-1-D2, 214C was extensively converted in the rat to labeled acetate which contained the 14C entirely in the carboxyl carbon, and had approximately the same ratio of 14 C D as the administered CD2OH· 14CH2NH2. In view of these findings the recently described pyridoxal phosphate dependent conversion of ethanolamine- O -phosphate to acetaldehyde, ammonia, and Pi, may best be interpreted as activation of hydrogen on the amino carbon atom by pyridoxal phosphate, followed by elimination of orthophosphate; hydrolysis of the resulting eneamine then yields acetaldehyde and ammonia (Fig. 1).

Purification of a terminal oxygenase in demethylation of C-30 of lanosterol

Abstract Oxygenation of the α-methyl group at C-4 (C-30) of lanosterol in solubilized rat liver microsomes was studied by 3 H release from [C-30- 3 H]-4, 4′-dimethylzymosterol. This activity required oxygen, a cyanide sensitive non-heme iron protein which was purified to homogeniety, either NADH or NADPH, and a labile fraction which has resisted resolution or purification. Attempts to implicate cytochrome P-450 reductase, cytochrome b 5 reductase, or cytochrome b 5 as electron transfer proteins in this system were not successful, suggesting that a new redox protein may be involved in oxygenation of C-30 of lanosterol.