Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit.
delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.
Srivastava
Alaka
A
Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
Beale
Samuel I
SI
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000622:Aminolevulinic Acid / Q000378:metabolism; D041883:Chlorobium / Q000201:enzymology*; D019281:Dimerization; D005990:Glycerol; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
D012346:RNA, Transfer, Amino Acyl; D006418:Heme; D000622:Aminolevulinic Acid; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase; D005990:Glycerol