X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: Insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

    Authors

    D. Apiyo; L. Zhao; M. D. Tsai;T. L. Selby

    Comments

    Authors: contact us about adding a copy of your work at STARS@ucf.edu

    Abbreviated Journal Title

    Biochemistry

    Keywords

    METAL-BINDING SITE; CRYSTAL-STRUCTURE; STREPTOMYCES-ANTIBIOTICUS; BACILLUS-CEREUS; DOMAIN; MECHANISM; STEREOSELECTIVITY; C-DELTA-1; RESIDUES; PROGRAM; Biochemistry & Molecular Biology

    Abstract

    Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thurigiensis to 2.1 angstrom resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca2+ ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.

    Journal Title

    Biochemistry

    Volume

    44

    Issue/Number

    30

    Publication Date

    1-1-2005

    Document Type

    Article

    Language

    English

    First Page

    9980

    Last Page

    9989

    WOS Identifier

    WOS:000230879900003

    ISSN

    0006-2960

    Share

    COinS