Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Biochemistry |
| Vol/bind | 38 |
| Udgave nummer | 10 |
| Sider (fra-til) | 2899-2908 |
| ISSN | 0006-2960 |
| DOI | |
| Status | Udgivet - 1999 |
Citationsformater
- APA
- Standard
- Harvard
- Vancouver
- Author
- BIBTEX
- RIS
I: Biochemistry, Bind 38, Nr. 10, 1999, s. 2899-2908.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
}
TY - JOUR
T1 - The Activity of Escherichia coli Dihydroorotate Dehydrogenase Is Dependent on a Conserved Loop Identified by Sequence Homology, Mutagenesis, and Limited Proteolysis
AU - Björnberg, Olof
AU - Grüner, Anne Charlotte
AU - Roepstorff, Peter
AU - Jensen, Kaj Frank
PY - 1999
Y1 - 1999
N2 - Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective with respect to substrate and product binding. Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed.
AB - Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate. The enzyme from Escherichia coli was overproduced and characterized in comparison with the dimeric Lactococcus lactis A enzyme, whose structure is known. The two enzymes represent two distinct evolutionary families of dihydroorotate dehydrogenases, but sedimentation in sucrose gradients suggests a dimeric structure also of the E. coli enzyme. Product inhibition showed that the E. coli enzyme, in contrast to the L. lactis enzyme, has separate binding sites for dihydroorotate and the electron acceptor. Trypsin readily cleaved the E. coli enzyme into two fragments of 182 and 154 residues, respectively. Cleavage reduced the activity more than 100-fold but left other molecular properties, including the heat stability, intact. The trypsin cleavage site, at R182, is positioned in a conserved region that, in the L. lactis enzyme, forms a loop where a cysteine residue is very critical for activity. In the corresponding position, the enzyme from E. coli has a serine residue. Mutagenesis of this residue (S175) to alanine or cysteine reduced the activities 10000- and 500-fold, respectively. The S175C mutant was also defective with respect to substrate and product binding. Structural and mechanistic differences between the two different families of dihydroorotate dehydrogenase are discussed.
U2 - 10.1021/bi982352c
DO - 10.1021/bi982352c
M3 - Journal article
SN - 0006-2960
VL - 38
SP - 2899
EP - 2908
JO - Biochemistry
JF - Biochemistry
IS - 10
ER -