HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 31, 28812-28822, August 1, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/31/28812    most recent M303767200v2 M303767200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nørager, S. Articles by Larsen, S. Search for Related Content PubMed PubMed Citation Articles by Nørager, S. Articles by Larsen, S. Social Bookmarking                      What's this?

Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function^*

Sofie Nørager , Susan Arent , Olof Björnberg , Mette Ottosen , Leila Lo Leggio , Kaj Frank Jensen  and Sine Larsen  ¶

From the Centre for Crystallographic Studies, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark and the Department of Biological Chemistry, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen, Denmark

Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis. The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis. We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site. Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.

Received for publication, April 10, 2003

The atomic coordinates and structure factors (codes 1JUB, K136E; 1OVD, K136E(Oro); 1JRB, P56A(Oro); 1JQX, R57A(Oro); and 1JRC, N67A(Oro)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Danish National Research Foundation, the Danish National Science Research Council, and the Carlsberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: European Synchrotron Radiation Facility, B.P. 220 Grenoble Cedex, France. Tel.: 45-35-32-0282; Fax: 45-35-32-0299; E-mail: sine{at}ccs.ki.ku.dk and slarsen{at}esrf.fr.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. P. Combe, J. Basran, P. Hothi, D. Leys, S. E. J. Rigby, A. W. Munro, and N. S. Scrutton Lys-D48 Is Required for Charge Stabilization, Rapid Flavin Reduction, and Internal Electron Transfer in the Catalytic Cycle of Dihydroorotate Dehydrogenase B of Lactococcus lactis J. Biol. Chem., June 30, 2006; 281(26): 17977 - 17988. [Abstract] [Full Text] [PDF]

				J. Shi, J. Dertouzos, A. Gafni, D. Steel, and B. A. Palfey Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis PNAS, April 11, 2006; 103(15): 5775 - 5780. [Abstract] [Full Text] [PDF]