Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli

Abstract

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.

Purification and partial characterisation of barley glutamyl‐tRNAGlu reductase, the enzyme that directs glutamate to chlorophyll biosynthesis

“…The E. coli hemA gene was cloned and sequenced simultaneously by three groups (Drolet et al, 1989;Li et al, 1989;Verkamp and Chelm, 1989). The study demonstrating the C5 pathway in E. coli also showed that Glu-tRNA synthetase and GSA-aminotransferase activities were present in the mutant strains, and that in vitro 5-aminolevulinic acid synthesis could be rkstored by addition of Chlorella vulgaris GlutRNA reductase to the mutant extracts (Avissar and Beale, 1989). Thus the 5-aminolevulinic acid auxotrophy in hemA mutants is caused by the absence of Glu-tRNA reductase activity.…”

“…5-aminolevulinic acid requiring mutants in E. coli were divided into two classes corresponding to mutations in two loci termed hemA (Sasarman et al, 1968) and hemW(popC) (Powell et al, 1973) both claimed to encode 5-aminolevulinic acid synthase. This apparent discrepancy was explained, when it was discovered that E. coli synthesizes 5-aminolevulinic acid from glutamate by the threestep C5 pathway (Avissar and Beale, 1989). The hemL gene was shown to encode GSA-aminotransferase (Grimm et al, 1991), while the function of the hem4 gene remained unclear.…”

“…The 5-aminolevulinic acid auxotroph hem4 mutant of E. coli lacks Glu-tRNA reductase activity (Avissar and Beale, 1989). It has been suggested that h e d encodes Glu-tRNA reductase, but overexpressed and partially purified hemA gene product shows no or very poor activity when compared to the purified Glu-tRNA reductases.…”

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

“…In one route, used by animals, yeast, and bacteria such as Rhodobacter and Rhizobium, a single enzyme condenses glycine with succinyl-CoA to produce 6- (3). Additionally, the conversion of glutamate to 6-aminolevulinate in extracts of the hemA mutant was restored in vitro by the addition of crude preparations of the reductase from Chlorella (4). Sequencing the first 18 amino acids from the N terminus of the reductase purified from barley chloroplasts provided the proof that the hemA gene codes for the glutamyl tRNAGlU reductase (5).…”

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

“…The E. coli hemA gene has been cloned by complementation of the original hemA strain by several groups (11,22,42), and homologous hemA genes have been isolated from S. typhimurium (13) and B. subtilis (33). Although direct biochemical evidence for the function of HemA has not been reported, reconstitution experiments with extracts of mutant strains implicates HemA at the level of Glu-tRNAG"u conversion to GSA by the Glu-tRNA reductase (3,4). The original E. coli popC mutants have perished (27a), but the hemL locus of S. typhimurium appears to be analogous (13,14).…”