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.
Duewel
H S
HS
Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, 48109-1065, USA.
Sheflyan
G Y
GY
Woodard
R W
RW
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008070:Lipopolysaccharides / Q000096:biosynthesis; D010428:Pentosephosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism
C121801:3-deoxy-manno-octulosonate-8-phosphate; D008070:Lipopolysaccharides; D010428:Pentosephosphates; D011994:Recombinant Proteins; D013400:Sugar Acids; D013403:Sugar Phosphates; C002532:2-keto-3-deoxyoctonate; C028055:arabinose 5-phosphate; D010728:Phosphoenolpyruvate; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases