Thermotoga maritima 3-Deoxy-D-arabino-heptulosonate 7-Phosphate (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 °C. The enzyme is extremely thermostable, with 50% of its initial activity retained after incubation for ∼5 h at 80 °C, 21 h at 70 °C, and 86 h at 60 °C. The enzyme appears to follow Michaelis-Menten kinetics with Km for phosphoenolpyruvate = 9.5–13 μm, Km for d-erythrose 4-phosphate = 57.3–350.1 μm, and kcat = 2.3–7.6 s–1 between 50 °C and 70 °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.

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