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The metal specificity of D-xylose isomerase from Streptomyces rubiginosus was examined by site-directed mutagenesis. The activation constants for metal ion (Mg^2+, Mn^2+, or Co^2+) of wild-type and mutant enzymes were determined by titrating the metal ion-free enzyme with Mg^2+, Mn^2+ , and Co^2+, respectively. Substitutions of amino acids either on coordinated or around the M2 site (His-220, Asn185, Glu-186, and Glu-221) dramatically affected the activation constants as well as activity. A decrease of metal binding affinity was most significant in the presence of Mg^2+. When compared with the wild-type enzymes, the binding affinity of H220S and N185K for Mg^2+ was decreased by 10-15-fold, while the affinity for Mn^2+, or Co^2+ only decreased by 3-5-fold. All the mutations close to the M2 site changed their metal preference from Mg^2+ to Mn^2+ or Co^2+. These altered metal preferences may be caused by a relatively weak binding affinity of Mg^2+ to the enzyme. Thermal inactivation studies of mutants at the M2 site also support the importance of the M2 site geometry for metal specificity as well as the thermostability of the enzyme. Mutations of other important groups hardly affected the metal preference, although pronounced effects on the kinetic parameters were sometimes observed. This study proposes that the metal specificity of D-xylose isomerase can be altered by the perturbation of the M2 site geometry, and that the different metal preference of Group Ⅰ and Group Ⅱ D-xylose isomerases may be caused by nonconserved amino acid residues around the M2 site.
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