Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme.
3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D-arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate. All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli, were believed not to require a metal cofactor for catalytic activity. However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme. Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes. One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori. In order to test this model, we have cloned the kdsa gene encoding H. pylori KDO-8-P synthase, and overexpressed and purified the protein. This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity. The Zn(2+) in the enzyme could be quantitatively replaced by Cd(2+), which increased the observed k(cat) by approximately 2-fold, and decreased the apparent K(m)(A-5-P) by approximately 6.5-fold. Furthermore, removal of the Zn(2+) from the enzyme did not greatly perturb its circular dichroism spectra. Thus, the divalent metal most likely serves as cofactor directly involved in catalysis.
Krosky
Daniel J
DJ
Infection Discovery, Cancer and Infection Research Area, AstraZeneca R&D Boston, Waltham, MA 02145, USA. dkrosky@jhmi.edu
Alm
Richard
R
Berg
Mikael
M
Carmel
Gilles
G
Tummino
Peter J
PJ
Xu
Bo
B
Yang
Wei
W
D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002104:Cadmium / Q000737:chemistry; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D010848:Picolinic Acids / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D015032:Zinc / Q000737:chemistry*
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010848:Picolinic Acids; D011994:Recombinant Proteins; D002104:Cadmium; D003035:Cobalt; D004492:Edetic Acid; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases; D015032:Zinc; C004860:dipicolinic acid