The escherchia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase

Journal Article · Sat Jun 01 04:00:00 EDT 1991 · Journal of Bacteriology; (United States)

OSTI ID:5570244

Ilag, L L; Jahn, D; Soell, D ^[1]; Eggertsson, G ^[2]

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1. Yale Univ., New Haven, CT (United States)
2. Univ. of Iceland, Reykjavik (Iceland)

[sigma]-Aminolevulinic acid (ALA), the first committed precursor of porphyrin biosynthesis, is formed in Escherichia coli by the C[sub 5] pathway in a three-step, tRNA-dependent transformation from glutamate. The first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase, are known in E. coli. Here we present the mapping and cloning of the gene for the third enzyme, glutamate 1-semialdehyde (GSA) aminotransferase, and an initial characterization of the purified enzyme, Ethylmethane sulfonate-induced mutants of E. coli AB354 which required ALA for growth were isolated by selection for respiration-defective strains resistent to the aminoglycoside antibiotic kanamycin. Two mutations were mapped to min 4 at a locus named hemL. Map positions and resulting phenotypes suggest that hemL may be identical with the earlier described porphyrin biosynthesis mutation popC. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding clone pLC4-43 of the Clarke-Carbon bank allowed the isolation of the gene. Physical mapping showed that hemL mapped clockwise next to fhuB. The hemL gene product was overexpressed and purified to apparent homogeneity. The pure protein efficiently converted GSA to ALA. The reaction was stimulated by the addition of pyridoxal 5[prime]-phosphate or pyridoxamine 5[prime]-phosphate and inhibited by gabaculine or aminooxyacetic acid. The molecular mass of the purified GSA aminotransferase under denaturing conditions was 40,000 Da, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an apparent native molecular mass of approximately 80,000 Da, as determined by rate zonal sedimentation on glycerol gradients and molecular sieving through Superose 12, which indicates a homodimeric [alpha][sub 2] structure of the protein.

• OSTI ID: 5570244
• Journal Information: Journal of Bacteriology; (United States), Journal Name: Journal of Bacteriology; (United States) Vol. 173:11; ISSN JOBAAY; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

550200 -- Biochemistry
550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINOTRANSFERASES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CLONING
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
MAPPING
MOLECULAR STRUCTURE
NITROGEN TRANSFERASES
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PORPHYRINS
PROTEINS
RNA
SYNTHESIS
TRANSFER RNA
TRANSFERASES