Journal of Biochemistry Advance Access originally published online on February 14, 2007
Journal of Biochemistry 2007 141(4):513-524; doi:10.1093/jb/mvm053
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© 2007 The Japanese Biochemical Society.
Human Xanthine Oxidase Changes its Substrate Specificity to Aldehyde Oxidase Type upon Mutation of Amino Acid Residues in the Active Site: Roles of Active Site Residues in Binding and Activation of Purine Substrate
1Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan; and 2Division of Kidney and Hypertension, Department of Internal Medicine, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan
*To whom correspondence should be addressed. Tel: +81-3-3822-2131, Fax: +81-3-5685-3054, E-mail: nishino{at}nms.ac.jp
Received December 28, 2006; Accepted January 28, 2007
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Abstract |
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Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 Å resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.
Key Words: xanthine oxidoreductase, aldehyde oxidoreductase, molybolenum cofactor, molybdenum, hydroxylase, human xanthine oxidase, allopurinol, xanthinuria
Abbreviations: AFR25, activity to flavin ratio; AO, aldehyde oxidase; KPB, potassium phosphate buffer; MoCo, molybdopterin cofactor; XDH, xanthine dehydrogenase; XO, xanthine oxidase; XOR, xanthine oxidoreductase
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