2Z28

Thr109Val dihydroorotase from E. coli

Summary for 2Z28

• Entry DOI: 10.2210/pdb2z28/pdb
• Related: 1XGE 2Z24 2Z25 2Z26 2Z27 2Z29 2Z2A 2Z2B
• Descriptor: Dihydroorotase, ZINC ION, (4S)-2,6-DIOXOHEXAHYDROPYRIMIDINE-4-CARBOXYLIC ACID, ... (5 entities in total)
• Functional Keywords: tim barrel, hydrolase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 78134.09

Authors

Lee, M.,Maher, M.J.,Guss, J.M. (deposition date: 2007-05-17, release date: 2007-10-09, Last modification date: 2023-11-15)

Primary citation

Lee, M.,Maher, M.J.,Christopherson, R.I.,Guss, J.M.
Kinetic and Structural Analysis of Mutant Escherichia coli Dihydroorotases: A Flexible Loop Stabilizes the Transition State
Biochemistry, 46:10538-10550, 2007

PubMed Abstract: Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.

PubMed: 17711307
DOI: 10.1021/bi701098e

Experimental method

X-RAY DIFFRACTION (1.87 Å)