Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme
| Autor: | Richard A. Alm, Mikael Berg, Bo Xu, Wei Yang, Peter J. Tummino, Daniel J Krosky, Gilles Carmel |
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| Rok vydání: | 2002 |
| Předmět: |
Stereochemistry
Biophysics chemistry.chemical_element Zinc medicine.disease_cause Biochemistry Cofactor Structural Biology Escherichia coli medicine Cloning Molecular Enzyme Inhibitors Picolinic Acids Molecular Biology Edetic Acid Aldehyde-Lyases Chelating Agents chemistry.chemical_classification Aquifex aeolicus Helicobacter pylori ATP synthase biology Chemistry Circular Dichroism Cobalt biology.organism_classification Recombinant Proteins carbohydrates (lipids) Kinetics Dicarboxylic acid Enzyme Spectrophotometry biology.protein lipids (amino acids peptides and proteins) Phosphoenolpyruvate carboxykinase Cadmium |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1594:297-306 |
| ISSN: | 0167-4838 |
| Popis: | 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 ∼2-fold, and decreased the apparent K m (A-5-P) by ∼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. |
| Databáze: | OpenAIRE |
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