Functional and biochemical characterization of a recombinant 3-Deoxy-D-manno-octulosonic acid 8-phosphate synthase from the hyperthermophilic bacterium Aquifex aeolicus.

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 (1)H 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 degrees C and with half-lives of 1.5 h (90 degrees C), 8.1 h (80 degrees C), and 30.3 h (70 degrees C). KdsA appeared to follow Michaelis-Menton kinetics with K(A5-P)(m) = 8 - 74 microM, K(PEP)(m) = 43-28 microM, and k(cat) = 0.4-2.0 s(-1) between 60 and 90 degrees C. Copyright 1999 Academic Press.