Structure of the C123S mutant of dienelactone hydrolase (DLH) bound with the PMS moiety of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF)

The structure of DLH (C123S) with PMS bound was solved to 2.5 Angstrom reso lution (R factor = 15.1%). PMSF in 2-propanol was delivered directly to cry stals in drops and unexpectedly caused the crystals to dissolve. New crysta ls displaying a different morphology emerged within 2 h in situ, a phenomen on that appears to be described for the first time. The changed crystal for m reflected altered crystal-packing arrangements elicited by structural cha nges to the DLH (C123S) molecule on binding inhibitor. The new unit cell re mained in the P2(1)2(1)2(1) space group but possessed different dimensions. The structure showed that PMS binding in DLH (C123S) caused conformational changes in the active site and in four regions of the polypeptide chain th at contain reverse turns. In the active site, residues with aromatic side c hains were repositioned in an edge-to-face cluster around the PMS phenyl ri ng. Their redistribution prevented restabilization of the triad His202 side chain, which was disordered in electron-density maps. Movements of other r esidues in the active site were shown to be related to the four displaced r egions of the polypeptide chain. Their implied synergy suggests that DLH ma y be able to accommodate and catalyse a range of compounds unrelated to the natural substrate owing to an inherent coordinated flexibility in its over all structure. Implications for mechanism and further engineering studies a re discussed.