Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

摘要: The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography site-directed mutagenesis after cloning overexpression in Escherichia coli. crystal wild-type enzyme has refined to an R factor 15.2% against diffraction data 2.2-A resolution. structures a number binary ternary complexes involving mutant enzymes, divalent cations Mg2+, Co2+, or Mn2+, either substrate analogs also determined comparable factors. Two metal sites are identified. Metal site 1 is four-coordinated tetrahedral absence six-coordinated octahedral its presence; O2 O4 atoms linear inhibitors bind 1. 2 all cases; position changes 0.7 A when it binds O1 more than O2; these bonds replace carboxylate ligands protein. Side chains involved binding substituted mutagenesis. biochemical properties enzymes presented. Together with structural data, they demonstrate that two ions play essential part substrates, stabilizing their open form, catalyzing hydride transfer between C1 C2 positions.