Cloning and characterization of adenylate kinase from Chlamydia pneumoniae

Chlamydiae proliferate only within the infected host cells and are thought to be "energy parasites," because they take up ATP from the host cell as an energy source. In the present study, we isolated from Chlamydia pneumoniae the gene encoding adenylate kinase (AK). Using the enzyme produced in Escherichia coli, its properties were characterized. K(m) values for AMP and for ADP of the purified C. pneumoniae AK (AKcpn) were each 330 microm, which is significantly higher than the reported values of other AKs, whereas K(m) for ATP was 24 microm, which was rather lower than others. AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein. Mass spectrometric analysis of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues in the LID domain of the enzyme. The apo-AKcpn that lost zinc retained AK activity, although K(m) for AMP of apo-AKcpn increased about 2-fold and V(max) decreased about one-half from that of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to trypsin digestion than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc. A mutated protein in which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity. The mutated protein was more sensitive to protease than the apo-AKcpn. These results indicate that zinc in AKcpn, although not essential for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore, the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.