Cloning, expression, and biochemical characterization of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase from the hyperthermophilic bacterium Aquifex pyrophilus.
3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by (1)H NMR analysis. KDO8P synthase showed maximum activity at 80 degrees C and pH 5.5-6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 degrees C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 degrees C were K(m)(A5P)=70 microM, K(m)(PEP)=290 microM, and k(cat)=4 s(-1). The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.
Shulami
Smadar
S
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Yaniv
Orit
O
Rabkin
Emilia
E
Shoham
Yuval
Y
Baasov
Timor
T
D000446:Aldehyde-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D008670:Metals; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases