ABSTRACT
The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5-1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b2, whereas the hitl mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.
Subject(s)
Fungal Proteins/metabolism , Pichia/metabolism , Riboflavin/biosynthesis , Aconitate Hydratase/metabolism , Genes, Fungal , Iron/metabolism , L-Lactate Dehydrogenase (Cytochrome)/metabolism , Mitochondria/metabolism , Pichia/genetics , Point MutationSubject(s)
Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Pichia/genetics , Riboflavin Synthase/genetics , Cloning, Molecular , DNA, Fungal/genetics , Genes, Fungal/genetics , Genetic Vectors , Mutation , Phenotype , Plasmids/genetics , Recombination, Genetic , Transformation, GeneticABSTRACT
Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB7 of this enzyme. Experiments showed that the presence of an ARS element and the promoter region of the gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondii are discussed.
Subject(s)
Pichia/genetics , Transformation, Genetic , GTP Cyclohydrolase/genetics , Mutation , Phenotype , Pichia/enzymology , Plasmids/genetics , Promoter Regions, GeneticABSTRACT
The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38,711 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type II from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiae. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits.
Subject(s)
GTP Cyclohydrolase/genetics , Genes, Fungal , Pichia/enzymology , Pichia/genetics , Riboflavin/biosynthesis , Amino Acid Sequence , Bacillus/enzymology , Bacillus/genetics , Base Sequence , Cloning, Molecular , DNA, Fungal/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Molecular Sequence Data , Pichia/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino AcidABSTRACT
Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.
Subject(s)
Bacillus subtilis/enzymology , GTP Cyclohydrolase/isolation & purification , Amino Acids/analysis , Cations , Chelating Agents , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , GTP Cyclohydrolase/antagonists & inhibitors , GTP Cyclohydrolase/metabolism , Hydrogen-Ion Concentration , Kinetics , MetalsABSTRACT
Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.
Subject(s)
Bacillus subtilis/enzymology , GTP Cyclohydrolase/genetics , Cloning, Molecular , DNA, Bacterial/genetics , Electrophoresis , Magnesium/metabolism , Manganese/metabolism , Operon , Plasmids , Protein Biosynthesis , Restriction Mapping , Riboflavin/geneticsABSTRACT
All the structural genes of riboflavin biosynthesis are shown to be located on the 2.8 MD DNA fragment, using the collection of plasmids, carrying the Bacillus subtilis riboflavin operon fragments and Bacillus subtilis strains, containing various deletions of rib-operon for analysis. The proximal Bgl II site is shown to be located between promoter P1 and the first structural gene ribG. The distal Hind III site of fragment C is the left bound of the rib-operon.
