The Extraordinary Specificity of Xanthine Phosphoribosyltransferase from Bacillus subtilis Elucidated by Reaction Kinetics, Ligand Binding, and Crystallography

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• 作者：Susan Arent ; Anders Kadziola ; Sine Larsen ; Jan Neuhard ; and Kaj Frank Jensen
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：May 30, 2006
• 年：2006
• 卷：45
• 期：21
• 页码：6615 - 6627
• 全文大小：509K
• 年卷期：v.45,no.21(May 30, 2006)
• ISSN：1520-4995

文摘

Xanthine phosphoribosyltransferase (XPRTase) from Bacillus subtilis is a representative ofthe highly xanthine specific XPRTases found in Gram-positive bacteria. These XPRTases constitute adistinct subclass of 6-oxopurine PRTases, which deviate strongly from the major class of H(X)GPRTaseswith respect to sequence, PRPP binding motif, and oligomeric structure. They are more related with thePurR repressor of Gram-positive bacteria, the adenine PRTase, and orotate PRTase. The catalytic functionand high specificity for xanthine of B. subtilis XPRTase were investigated by ligand binding studies andreaction kinetics as a function of pH with xanthine, hypoxanthine, and guanine as substrates. The crystalstructure of the dimeric XPRTase-GMP complex was determined to 2.05 Å resolution. In a sequentialreaction mechanism XPRTase binds first PRPP, stabilizing its active dimeric form, and subsequentlyxanthine. The XPRTase is able also to react with guanine and hypoxanthine albeit at much lower (10^-4-fold) catalytic efficiency. Different pK_a values for the bases and variations in their electrostatic potentialcan account for these catalytic differences. The unique base specificity of XPRTase has been related toa few key residues in the active site. Asn27 can in different orientations form hydrogen bonds to anamino group or an oxo group at the 2-position of the purine base, and Lys156 is positioned to make ahydrogen bond with N7. This and the absence of a catalytic carboxylate group near the N7-position requirethe purine base to dissociate a proton spontaneously in order to undergo catalysis.