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 Esch erichia coil, its properties were characterized. K-m values for AMP and for ADP of the purified C, pneumoniae AK (AKcpn) were each 330 muM, which is s ignificantly higher than the reported values of other AKs, whereas K-m for ATP was 24 muM, 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 i s associated with four cysteine residues in the LID domain of the enzyme. T he apo-AKcpn that lost zinc retained AK activity, although K-m for AMP of a po-AKcpn increased about 2-fold and V-max decreased about one-half from tha t of holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to tryps in digestion than the holo-AKcpn. Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was de pendent on zinc. A mutated protein in which cysteine residues in the LID do main were substituted by other amino acids lost both zinc and enzyme activi ty. The mutated protein was more sensitive to protease than the apo AKcpn. These results indicate that zinc in AKcpn, although not essential for the c atalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein. Furthermore, the catalytic properties of AKcpn sug gest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.