FAS Abstracts 2004 Meeting Page

Environmental Chemistry & Chemical Sciences A (ENV): ENV-2

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Structural studies of an engineered calcium-binding bacterial phospholipase-C.  D.O. APIYO (1) and T.L. SELBY (1,2).  Dept. of Chemistry (1) and Biomolecular Sciences Program (2), Univ. of Central Florida, Orlando, FL 32816.  Metal ions play significant roles in many biological systems. The metal may act as a nucleation site from which the polypeptide folds, thus imparting structural stabilization to the enzyme or it may instead be responsible for the enzyme’s thermodynamic stability coupled to its role in enzymic function. Metal binding sites are therefore attractive targets in protein engineering.  The catalytic active-site of a bacterial phospatidyl calcium independent phospholipase C was recently modified to create a calcium binding enzyme (R69D) in an effort to understand the catalytic role of calcium in signal transduction. X-ray crystallographic studies of the engineered enzyme reveal that the bound calcium occupies a similar position as the guanidinium side chain (involved in catalysis in bacterial phospholipase Cs) of the arginine in the wild type enzyme. The calcium is ligated to two protein ligands (Asp37 and Asp73 corresponding to Asp33 and Arg69 in the wild type) and a water molecule from the solvent. This results into a distorted tetrahedral geometry which differs remarkably from the octahedral geometry observed in the calcium-dependent mammalian phospholipase C’s. Interestingly, the two catalytic histidines (His36 and His86) both have their conformations significantly altered (from the wild type) upon calcium-binding, with their imidazole side-chains oriented toward the metal. In contrast, the catalytic ligands of the calcium-dependent mammalian phospholipase Cs are distanced much further away from the calcium and have an outward orientation of their imidazole side chain.  It is apparent that the calcium in the engineered enzyme is involved in local structural stability that may result into a change in mechanism of catalysis for bacterial phospholipase Cs.