MECHANISM OF THE REACTION CATALYZED BY MANDELATE RACEMASE .2. CRYSTAL-STRUCTURE OF MANDELATE RACEMASE AT 2.5-A RESOLUTION - IDENTIFICATION OF THE ACTIVE-SITE AND POSSIBLE CATALYTIC RESIDUES

The crystal structure of mandelate racemase (MR) has been solved at 3.0-angstrom resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-angstrom resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta-topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eighth alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-angstrom resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts. The results of these structural studies support the conclusions reached from the chemical and kinetic experiments described in the preceding paper regarding the number and chemical properties of the bases in the active site of MR [Powers, V. M., Koo, C., Kenyon, G. K., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue)]. This is the first three-dimensional structure of a racemase to be determined.