1-deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

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 isopr enoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phospha te. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two ope n reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-ph osphate synthase. ORF 2816 is located in the photosynthesis-related gene cl uster, along with most of the genes required for synthesis of the photosynt hetic 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-xylulo se 5-phosphate synthase A and B, containing a His, 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 syntha se A and B: K-m(pyruvate) = 0.61 and 3.0 mM, K-m(D-glyceraldehyde 3-phospha te) = 150 and 120 muM, and V-max = 1.9 and 1.4 mu mol/min/mg in 200 mM sodi um citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate syntha se B complemented the disrupted essential drs gene in E. coli strain FH11.