Molecular biological study of early steps of heme biosynthesis in Escherichia coli
The heme molecule is the prosthetic group of hemoglobins, cytochromes, catalases and peroxidases, and modified tetrapyrroles are the active moieties of the chlorophylls and vitamin B{sub 12}. The pathway of heme biosynthesis is highly conserved except for the formation of 5-aminolevulinic acid (ALA), the first common intermediate. ALA can be synthesized from glutamic acid (C{sub 5} pathway) or succinyl CoA and glycine (C{sub 4} pathway). In E. coli, the heme biosynthetic pathway consists of more than eight enzymatic steps. The genes encoding these enzymes are widely scattered on the chromosome. An E. coli heme-requiring, hemin-permeable mutant had no detectable 5-aminolevulinic acid dehydratase (ALA D) or porphobilinogen deaminase (PBG D) activities. The gene which complemented this mutation was cloned. PBG D activity was restored to normal levels, but the activity of ALA D was 20-30 fold higher than normal. A maxicell procedure confirmed that the cloned gene was hemB. The hemB gene was sequenced. Two promoter regions, two Shine-Dalgarno sequences and two possible initiation sites were identified. Extensive homologies with yeast (36%), human liver (40%) and rat liver (40%) amino acid sequences were observed, especially in the sixteen-amino acid Zn-binding region (75%) and the four amino acids surrounding the essential lysine at the active site (100% for rate and human proteins). Analysis of promoter strength and two independent analyses of codon usage indicated that the hemB gene is moderately-expressed. E. coli hemA gene was cloned and sequenced. Complemented mutants overproduced 5-ALA and porphyrins. The cloned sequence appears to encode a 46 kDa protein. The amino acid sequence of the cloned gene product showed no significant homologies with any cloned ALA synthase nor with any protein in two data banks.
- Research Organization:
- City Univ. of New York, NY (USA)
- OSTI ID:
- 5526907
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ESCHERICHIA COLI
BIOCHEMISTRY
HEME
BIOSYNTHESIS
HYDRO-LYASES
GENES
AMINO ACID SEQUENCE
BIOLOGICAL PATHWAYS
CARBON 14 COMPOUNDS
CHROMOSOMES
CLONING
ENZYME ACTIVITY
HYDROLASES
MUTANTS
TRACER TECHNIQUES
BACTERIA
CARBON-OXYGEN LYASES
CARBOXYLIC ACIDS
CHEMISTRY
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYASES
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
PORPHYRINS
SYNTHESIS
550201* - Biochemistry- Tracer Techniques