W0044

One Domain Helps to Solve Another in Allosteric PLP-Dependent Threonine Deaminase. Travis Gallagher, Edward Eisenstein and Gary L. Gilliland; Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850 USA

Biosynthetic threonine deaminase is an allosteric tetramer of MW 220 Kdal regulating the synthesis of the branched chain amino acids in E. coli. We have analyzed by diffraction several crystal forms of this enzyme, including two distinct forms that grow under identical conditions. The best diffracting crystals (2.3 Å native data) belong to space group I222, where the asymmetric unit contains one 514-residue chain and one PLP group. The structure has been determined in this space group, and has been refined to R=0.20 and Rfree=0.34, revealing details of PLP binding in the active site and the relation of the catalytic and regulatory domains. Implications of structural and biophysical data for the allosteric mechanism will be discussed.

Structure determination was by SIRAS and also utilized a novel technique wherein the confidently-fit catalytic domain was used to improve phases and thus maps of the more difficult regulatory domain. We call the technique IMR (Iterative Map Recombination) because it involves iteratively adding structure factors for the two domains, in conjunction with standard phase combination techniques, to improve the estimate of total phase. A similar method was used in a previous structure determination where molecular replacement located a known domain, but an unknown domain was too large to be revealed by simple or weighted difference Fourier methods.