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Journal of Bacteriology, January 2001, p. 1-11, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.1-11.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

1-Deoxy-D-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

Frederick M. Hahn,dagger Lisa M. Eubanks, Charles A. Testa, Brian S. J. Blagg, Jonathan A. Baker, and C. Dale Poulter*

Department of Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 22 May 2000/Accepted 24 August 2000

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His6 tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: Kmpyruvate = 0.61 and 3.0 mM, KmD-glyceraldehyde 3-phosphate = 150 and 120 µM, and Vmax = 1.9 and 1.4 µmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.

* Corresponding author. Mailing address: Department of Chemistry, University of Utah, Salt Lake City, UT 84112. Phone: (801) 581-6685. Fax: (801) 581-4391. E-mail: poulter{at}chemistry.utah.edu.

dagger Present address: Maui Agricultural Research Center, University of Hawaii, Kula, HI 96790.

Journal of Bacteriology, January 2001, p. 1-11, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.1-11.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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