Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase

Abstract

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.

Expression of the Soybean (Glycine max) Glutamate 1-Semialdehyde Aminotransferase Gene in Symbiotic Root Nodules

“…In addition, a termination codon was identified upstream of the open reading frame and in the same reading frame, showing that the entire coding region was present in the cloned cDNA. The putative protein shared 79% identity with the GSA aminotransferase expressed in barley leaves (Grimm, 1990; with bacterial aminotransferases (Elliott et al, 1990;Grimm et al, 1991). This identity, along with the observed complementation of the GSA aminotransferase structural gene mutant of E. coli (Table 11), strongly suggests that the cloned cDNA encodes a soybean GSA aminotransferase.…”

Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis.

“…Like other aminotransferases, GSA-AM utilizes pyridoxal phosphate as a cofactor. Sequence information is available for GSA-AM genes from E. coli (Grimm et al, 1991), 6. subrilis (Hansson et al, 1991), S. typhimurium (Elliott et al, 1990), Synechococcus (Grimm et al, 1991), barley (Grimm, 1990), and soybean (Sangwan and OBrian, 1993). Comparison of the deduced amino acid sequences revealed that GSA-AM proteins are very similar to one another; e.g., the Synechococcus and barley enzymes are 70% identical.…”

Characterization of δ-Aminolevulinic Acid Formation in Soybean Root Nodules

“…Each lane was cut into 1-cm strips and put into scintillation vials containing fluor and counted. Unlabeled ALA-pyrrole standard was also chromatographed as described above, and was visualized with Ehrlich spray reagent (7). The chromatographic profile of [3H]ALA-pyrrole was qualitatively the same as the 14C compound, but the tritium was quenched 8-to 10-fold by the paper, and thus those data are not shown.…”

“…Paper Chromatography of ALA-Pyrrole ['4C]ALA-pyrrole synthesized from U-["'C]glutamate and isolated as described above was chromatographed as described by Elliott et al (7). ALA-pyrrole in ether was applied to Whatman 3MM paper and chromatographed in a solvent containing n-butanol:n-proponal:5% (w/v) aqueous NH40H (2:1:1, v/v).…”