Thermotoga maritima 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase - The ancestral eubacterial DAHP synthase?

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 degreesC. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for similar to5 h at 80 degreesC, 21 h at 70 degreesC, and 86 h at 60 degreesC. The enzyme appears to follow Michaelis-Menten kinetics with K-m for phosphoenolpyruvate = 9.5-13 muM, K-m for D-erythrose 4-phosphate = 57.3-350.1 muM, and k(cat) = 2.3-7.6 s(-1) between 50 degreesC and 70 degreesC. 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+, Cd-2+, 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.