2z2b
From Proteopedia
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'''Deletion 107-116 mutant of dihydroorotase from E. coli''' | '''Deletion 107-116 mutant of dihydroorotase from E. coli''' | ||
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[[Category: Lee, M.]] | [[Category: Lee, M.]] | ||
[[Category: Maher, M J.]] | [[Category: Maher, M J.]] | ||
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 19:51:47 2008'' | |
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Revision as of 16:51, 4 May 2008
Deletion 107-116 mutant of dihydroorotase from E. coli
Overview
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.
About this Structure
2Z2B is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state., Lee M, Maher MJ, Christopherson RI, Guss JM, Biochemistry. 2007 Sep 18;46(37):10538-50. Epub 2007 Aug 21. PMID:17711307 Page seeded by OCA on Sun May 4 19:51:47 2008
