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Journal of Bacteriology, July 2005, p. 4444-4450, Vol. 187, No. 13
0021-9193/05/$08.00+0     doi:10.1128/JB.187.13.4444-4450.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit

Alaka Srivastava{dagger} and Samuel I. Beale*

Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

Received 12 December 2004/ Accepted 10 March 2005

{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 His6 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 His6-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNAGlu 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 ß-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.

* Corresponding author. Mailing address: Biomed, Box G-J4, Brown University, Providence, RI 02912. Phone: (401) 863-3129. Fax: (401) 863-1182. E-mail: sib{at}brown.edu.

{dagger} Present address: Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14642.

Journal of Bacteriology, July 2005, p. 4444-4450, Vol. 187, No. 13
0021-9193/05/$08.00+0     doi:10.1128/JB.187.13.4444-4450.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Nogaj, L. A., Srivastava, A., van Lis, R., Beale, S. I. (2005). Cellular Levels of Glutamyl-tRNA Reductase and Glutamate-1-Semialdehyde Aminotransferase Do Not Control Chlorophyll Synthesis in Chlamydomonas reinhardtii. Plant Physiol. 139: 389-396 [Abstract] [Full Text]  


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