Aspects of the regulation of heme biosynthesis in Escherichia coli K-12.
Item
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Title
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Aspects of the regulation of heme biosynthesis in Escherichia coli K-12.
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Identifier
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AAI9108185
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identifier
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9108185
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Creator
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Umanoff, Heywood.
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Contributor
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Advisers: Charlotte Russell | Sharon Cosloy
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Date
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1990
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Health Sciences, Medicine and Surgery
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Abstract
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Hemin-permeable mutants were isolated from hemA (glutamyl-tRNA dehydrogenase) and hemB (5-aminolevulinic acid dehydratase) mutants by nitrosoguanidine mutagenesis and selection on hemin-containing and unsupplemented media. When grown in hemin-containing media neither mutant exhibited porphobilinogen deaminase (PBG D) (hemC gene product) activity in crude extracts. Hemin-permeable prototrophs were obtained by complementation of the hemA and hemB mutant alleles with a phasmid transducing system. Complementation of each mutant allele with its corresponding functional gene resulted in restoration of PBG D activity in the extract. It was determined that the presence of porphobilinogen (PBG) was required for PBG D activity.;PBG D was partially purified from an overproducing E. coli strain. Incubation of this enzyme sample with either 10% formic acid or 1 M HCl resulted in the formation of porphyrins from enzyme-bound pyrroles, confirming earlier reports (Jordan and Warren, 1987). Incubation of the enzyme with p-hydroxymercuribenzoate, a known inhibitor of PBG D, did not release a novel dipyrromethane cofactor from the enzyme.;Hemin did not affect ALA synthesis when added to growing cultures of either a hemin-permeable hemB mutant or a hemin-permeable prototroph. Nor did it affect ALA D or PBG D activities in the extract, when hemin was added to growing cultures of various hemin-permeable mutants. Hemin did not affect the activity of partially-purified PBG D.;Anaerobic growth of C600 in rich media resulted in a marginal increase in PBG D activity but had no affect on ALA D activity. Glucose decreased PBG D activity by approximately two-fold when added to aerobically growing cultures of C600, but had no affect on ALA D activity.;The effects of the heme pathway-related metabolites, L-glutamatic acid, 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX, and hemin, on the in vitro transcription-translation of the E. coli hemA, hemB and hemC genes was studied. None of these compounds affected gene expression.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
