Skip Navigation


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
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
141/4/513    most recent
mvm053v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Nishino, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Nishino, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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

Yuichiro Yamaguchi1,2, Tomohiro Matsumura1, Kimiyoshi Ichida2, Ken Okamoto1 and Takeshi Nishino1,*

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


  Abstract

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


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
J. F. Alfaro, C. A. Joswig-Jones, W. Ouyang, J. Nichols, G. J. Crouch, and J. P. Jones
Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli
Drug Metab. Dispos., December 1, 2009; 37(12): 2393 - 2398.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. M. Pauff, H. Cao, and R. Hille
Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase: CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE
J. Biol. Chem., March 27, 2009; 284(13): 8760 - 8767.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
U. Dietzel, J. Kuper, J. A. Doebbler, A. Schulte, J. J. Truglio, S. Leimkuhler, and C. Kisker
Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase
J. Biol. Chem., March 27, 2009; 284(13): 8768 - 8776.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. M. Pauff, J. Zhang, C. E. Bell, and R. Hille
Substrate Orientation in Xanthine Oxidase: CRYSTAL STRUCTURE OF ENZYME IN REACTION WITH 2-HYDROXY-6-METHYLPURINE
J. Biol. Chem., February 22, 2008; 283(8): 4818 - 4824.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. M. Pauff, C. F. Hemann, N. Junemann, S. Leimkuhler, and R. Hille
The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus
J. Biol. Chem., April 27, 2007; 282(17): 12785 - 12790.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.