IDENTIFICATION OF A THIAMIN-DEPENDENT SYNTHASE IN ESCHERICHIA-COLI REQUIRED FOR THE FORMATION OF THE 1-DEOXY-D-XYLULOSE 5-PHOSPHATE PRECURSOR TO ISOPRENOIDS, THIAMIN, AND PYRIDOXOL

In Escherichia coli, 1-deoxy-D-xylulose (or its 5-phosphate, DXP) is t he biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenossische Technische Hochschule, Zurich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., S ayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187-1188], Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP syntha se) that catalyzes a thiamin diphosphate-dependent acyloin condensatio n reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-ph osphate to yield DXP, We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 un it/mg of protein, The reaction catalyzed by DXP synthase yielded exclu sively DXP, which was characterized by H-1 and P-31 NMR spectroscopy, Although DXP synthase of E. coli shows sequence similarity to both tra nsketolases and the E1 subunit of pyruvate dehydrogenase, it is a memb er of a distinct protein family, and putative DXP synthase sequences a ppear to be widespread in bacteria and plant chloroplasts.