Abstract
The kdsA gene from the hyperthermophilic bacterium Aquifex aeolicus was cloned into a vector for expression in Escherichia coli and the kdsA gene product, 3-deoxy-d-manno-octulosonic acid 8-phosphate synthase (KdsA), was overexpressed under optimized growth conditions. The thermophilic KdsA was purified using an efficient purification procedure including a heat-treatment step. Purified KdsA was shown to catalyze the formation of 3-deoxy-d-manno-octulosonic acid 8-phosphate from phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A 5-P) as determined from 1H NMR analysis of the product. Analytical gel filtration analysis indicated the native enzyme to be oligomeric. KdsA was extremely thermostable, exhibiting maximal activity at 95°C and with half-lives of 1.5 h (90°C), 8.1 h (80°C), and 30.3 h (70°C). KdsA appeared to follow Michaelis–Menton kinetics with KA5-Pm = 8 − 74 μM, KPEPm = 43–28 μM, and kcat = 0.4–2.0 s−1 between 60 and 90°C.
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