Molecular biological study of early steps of heme biosynthesis in Escherichia coli

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5526907

Li, Jianming

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
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
BACTERIA
BIOCHEMISTRY
BIOLOGICAL PATHWAYS
BIOSYNTHESIS
CARBON 14 COMPOUNDS
CARBON-OXYGEN LYASES
CARBOXYLIC ACIDS
CHEMISTRY
CHROMOSOMES
CLONING
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GENES
HEME
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDRO-LYASES
HYDROLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYASES
MICROORGANISMS
MOLECULAR STRUCTURE
MUTANTS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
PORPHYRINS
SYNTHESIS
TRACER TECHNIQUES