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Characterization of the novel Trypanosoma brucei inosine 5′-monophosphate dehydrogenase

Published online by Cambridge University Press:  01 February 2013

TOMOAKI BESSHO
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
SHOKO MORII
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
TOSHIHIDE KUSUMOTO
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
TAKAHIRO SHINOHARA
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
MASANORI NODA
Affiliation:
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
SUSUMU UCHIYAMA
Affiliation:
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
SATOSHI SHUTO
Affiliation:
Laboratory of Organic Chemistry for Drug Development, Faculty of Pharmaceutical Sciences, Hokkaido University, Nishi 6, Kita 12, Kita-ku, Sapporo 060-0812, Japan
SHIGENORI NISHIMURA
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
APPOLINAIRE DJIKENG
Affiliation:
Biosciences eastern and central Africa (BecA) Hub at the International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya The J Craig Venter Institute (JCVI), Rockville, MD 20876, USA
MICHAEL DUSZENKO
Affiliation:
Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Straße 4, 72076 Tübingen, Germany
SAMUEL K. MARTIN
Affiliation:
Retired from United States Army Medical Research Unit-Kenya, Unit 64109, APO AE 09831-64109
TAKASHI INUI*
Affiliation:
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
KILUNGA B. KUBATA*
Affiliation:
AU/NEPAD Agency Regional Office for Eastern and Central Africa, P.O. BOX 13601-00800, Nairobi, Kenya
*
*Corresponding authors: Dr Takashi Inui, Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Tel: +81-72-254-9473, Fax: +81-72-254-9921. E-mail: inuit@bioinfo.osakafu-u.ac.jp
*Fr Kilunga B. Kubata, AU/NEPAD Agency Regional Office for Eastern and Central Africa, P.O. BOX 13601-00800, Nairobi, Kenya. Tel: +254 733 665210/ +254 737 966687. E-mail: bkkubata@nepadst.org / brunokubata@yahoo.com

Summary

There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5′-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated Km values of 30 and 1300 μm for IMP and NAD, respectively. The obtained Km value of TbIMPDH for NAD is approximately 20–200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show Ki values of 3·2 μm, 21 nM and 3·3 nM for ribavirin 5′-monophosphate, mycophenolic acid and mizoribine 5′-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2013

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