The crystal structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design

Thymidylate synthase from Pneumocystis carinii (PcTS) is an especially impo rtant drug target, since P. carinii is a fungus that causes opportunistic p neumonia infections in immune-compromised patients and is among the leading causes of death of AIDS patients. Thymidylate synthase (TS) is the sole en zyme responsible for the de novo production of deoxythymidine monophosphate and hence is crucial for DNA replication in every organism. Inhibitors sel ective for P. carinii TS over human TS would be greatly beneficial in comba ting this disease. The crystal structure of TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.6 Angst rom resolution. A comparison with other species of TS shows that the volume of the closed PcTS active-site is 20 % larger than that of five other TS c losed active-sites. A two-residue proline insert that is strictly conserved among all fungal species of TS, and a novel C-terminal closing interaction involving a P. carinii-specific tyrosine residue are primarily responsible for this increase in volume. The structure suggests several options for de signing an inhibitor specific to PcTS and avoiding interactions with human TS. Taking advantage of the residue substitutions of P. carinii TS over hum an TS enables the design of a selective inhibitor. Additionally, the larger volume of the active-site of PcTS is an important advantage for designing de novo inhibitors that will exclude the human TS active-site through steri c hindrance. (C) 2000 Academic Press.