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Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination.
Biochim Biophys Acta. 2010 Dec; 1804(12):2198-206.BB

Abstract

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.

Authors+Show Affiliations

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20831907

Citation

Lohkamp, Bernhard, et al. "Insights Into the Mechanism of Dihydropyrimidine Dehydrogenase From Site-directed Mutagenesis Targeting the Active Site Loop and Redox Cofactor Coordination." Biochimica Et Biophysica Acta, vol. 1804, no. 12, 2010, pp. 2198-206.
Lohkamp B, Voevodskaya N, Lindqvist Y, et al. Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination. Biochim Biophys Acta. 2010;1804(12):2198-206.
Lohkamp, B., Voevodskaya, N., Lindqvist, Y., & Dobritzsch, D. (2010). Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination. Biochimica Et Biophysica Acta, 1804(12), 2198-206. https://doi.org/10.1016/j.bbapap.2010.08.014
Lohkamp B, et al. Insights Into the Mechanism of Dihydropyrimidine Dehydrogenase From Site-directed Mutagenesis Targeting the Active Site Loop and Redox Cofactor Coordination. Biochim Biophys Acta. 2010;1804(12):2198-206. PubMed PMID: 20831907.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination. AU - Lohkamp,Bernhard, AU - Voevodskaya,Nina, AU - Lindqvist,Ylva, AU - Dobritzsch,Doreen, Y1 - 2010/09/08/ PY - 2010/06/03/received PY - 2010/08/13/revised PY - 2010/08/31/accepted PY - 2010/9/14/entrez PY - 2010/9/14/pubmed PY - 2011/3/9/medline SP - 2198 EP - 206 JF - Biochimica et biophysica acta JO - Biochim Biophys Acta VL - 1804 IS - 12 N2 - In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding. SN - 0006-3002 UR - https://www.unboundmedicine.com/medline/citation/20831907/Insights_into_the_mechanism_of_dihydropyrimidine_dehydrogenase_from_site_directed_mutagenesis_targeting_the_active_site_loop_and_redox_cofactor_coordination_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-9639(10)00238-4 DB - PRIME DP - Unbound Medicine ER -