E. coli Dihydroorotate Dehydrogenase Reveals Structural and Functional Distinctions between Different Classes of Dihydroorotate Dehydrogenases

Nørager, S.; Jensen, Kaj Frank; Björnberg, Olof; Larsen, Sine

doi:10.1016/s0969-2126(02)00831-6

Cited by 99 publications

(137 citation statements)

References 43 publications

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“…With the active site loop in the open conformation these direct interactions are not possible, but they occur via a water molecule and Ser-129 O␥ is instead hydrogen-bonded to Gln-138 O⑀1. From the comparison with the DHODB and DHODC structures, we have proposed that this serine and Gln-138 in DHODA act as hinges of the active site loop (17). The exchange of the Ser-129 with alanine leads to the loss of the hydrogen bond formed by the side chain and must affect the loop movement and orotate binding site.…”

Section: Crystal Structuresmentioning

confidence: 99%

“…DHODA, DHODB, and DHODC (17) showed that Lys-213 only is conserved among the class 1a enzymes and that the open conformation seen in the K213E(Oro) structure corresponds to the conformation of the loop observed in the structure of the uncomplexed class 1B enzyme, DHODB (10). In the DHODB structure in complex with orotate the loop is in a closed conformation, but could only be traced partially, while in the open conformation it could be traced completely, indicating that it moves to carry out the reaction but is more stable in the open conformation.…”

Section: Roles Of Different Residues In Dhoda-mentioning

confidence: 99%

“…The class 2 DHODs are monomeric enzymes; there are structures known for the human and Escherichia coli DHOD (DHODC) (16,17). Relative to the class 1 enzymes, they possess an extended N terminus, which plays a role in the membrane association of the enzyme and provides the binding site for the respiratory quinones that serve as physiological electron acceptors (16,17).The earliest studies on DHOD from bovine liver (18) and Crithidia fasciculata (19) together with more recent studies on DHODB from Enterococcus faecalis (20) and on DHODC from E. coli (21) describe the stereospecific oxidation of (S)-dihydroorotate to orotate as mediated by an active site base. The active site base abstracts the C5-S proton of dihydroorotate (DHO) followed by hydride transfer from the C6 position of DHO to the N5 of FMN.…”

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confidence: 99%

“…The PyrK subunits each contain FAD and an [2Fe-2S] cluster, and the presence of these subunits enables the tetrameric enzyme to use NAD ϩ as electron acceptor (10,15). The class 2 DHODs are monomeric enzymes; there are structures known for the human and Escherichia coli DHOD (DHODC) (16,17). Relative to the class 1 enzymes, they possess an extended N terminus, which plays a role in the membrane association of the enzyme and provides the binding site for the respiratory quinones that serve as physiological electron acceptors (16,17).…”

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confidence: 99%

“…The active site base abstracts the C5-S proton of dihydroorotate (DHO) followed by hydride transfer from the C6 position of DHO to the N5 of FMN. Based on structural comparisons of class 1 and class 2 enzymes we have suggested that the mechanism for the first half-reaction is highly conserved, and two highly conserved lysines are essential for the catalytic function (17). In the class 1 enzymes the active site base is a cysteine residue (7) while in class 2 enzymes it is a serine (14).…”

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confidence: 99%

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

Nørager

Arent

Björnberg

et al. 2003

Journal of Biological Chemistry

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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.Dihydroorotate dehydrogenases (DHODs) 1 catalyze the stereospecific oxidation of (S)-dihydroorotate to orotate through reduction of their prosthetic FMN group. This is the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. In rapidly proliferating cells pyrimidine salvage pathways cannot compensate for the lack of UMP caused by inhibition of DHOD. This makes DHODs attractive targets for antiproliferative, antiparasitic, and immunosuppresive drugs used in organ transplantation and in treatment of inflammatory diseases (1-6).Based on presently known sequences, DHODs can be divided into two main classes (7). Class 1 enzymes, which are subdivided into class 1A and 1B enzymes, are cytosolic proteins, whereas class 2 enzymes are membrane-associated. The bacterium Lactococcus lactis contains genes that encode DHODs representing subclass 1A and 1B, DHODA, and DHODB. Crystal structures have been determined for both of these without and in the presence of the product orotate (8 -10). DHODA is a dimer formed by two identical PyrD subunits each containing an FMN group (11). The natural electron acceptor for DHODA is fumarate (12), but all DHODs can to some extent use a variety of other electron acceptors such as soluble quinones, dyes, and molecular oxygen (13, 14). For DHODA it has been suggested that the substrate and the natural electron acceptor use the same binding site. Kinetic investigations supported a one-site ping-pong mechanism and showed the second halfreaction to be the rate-limiting step for DHODA (13).The class 1B enzyme is a heterotetramer consisting of two PyrDB subunits, homologous to the PyrDA subunits of DHODA, and two PyrK subunits (15). The PyrK subunits each contain FAD and an [2Fe-2S] cluster, and...

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Section: Crystal Structuresmentioning

confidence: 99%

Section: Roles Of Different Residues In Dhoda-mentioning

confidence: 99%

mentioning

confidence: 99%