The gene encoding the 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase from the thermophilic microorganism Thermotoga maritima was cloned, and the enzyme was overexpressed in Escherichia coli. The purified DAHP synthase displays a homotetrameric structure and exhibits maximal activity at 90 degrees C. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for approximately 5 h at 80 degrees C, 21 h at 70 degrees C, and 86 h at 60 degrees C. The enzyme appears to follow Michaelis-Menten kinetics with Km for phosphoenolpyruvate = 9.5-13 microm, Km for d-erythrose 4-phosphate = 57.3-350.1 microm, and kcat = 2.3-7.6 s-1 between 50 degrees C and 70 degrees C. Metal analysis indicates that DAHP synthase as isolated contains Zn2+, and the enzyme is inactivated by treatment with EDTA. The apo-enzyme is partially reactivated by a variety of divalent metals including Zn2+, Cd2+, Mn2+, Cu2+, Co2+, and Ni2+. These observations suggest that T. maritima DAHP synthase is a metalloenzyme. The activity of T. maritima DAHP synthase is inhibited by two of the three aromatic amino acids (l-Phe and l-Tyr) formed in the Shikimate pathway. This report is the first description of a thermophilic eubacterial DAHP synthase.