Structures of competitive inhibitor complexes of protocatechuate 3,4-dioxygenase: multiple exogenous ligand binding orientations within the active site.
Protocatechuate 3,4-dioxygenase (3,4-PCD) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate to produce beta-carboxy-cis, cis-muconate. Crystal structures of Pseudomonas putida3,4-PCD [quaternary structure of (alphabetaFe3+)12] complexed with seven competitive inhibitors [3-hydroxyphenylacetate (MHP), 4-hydroxyphenylacetate (PHP), 3-hydroxybenzoate (MHB), 4-hydroxybenzoate (PHB), 3-fluoro-4-hydroxybenzoate (FHB), 3-chloro-4-hydroxybenzoate (CHB), and 3-iodo-4-hydroxybenzoate (IHB)] are reported at 2.0-2.2 A resolution with R-factors of 0. 0.159-0.179. The inhibitors bind in a narrow active site crevasse lined with residues that provide a microenvironment that closely matches the chemical characteristics of the inhibitors. This results in as little as 20% solvent-exposed surface area for the higher-affinity inhibitors (PHB, CHB, and FHB). In uncomplexed 3,4-PCD, the active site Fe3+ is bound at the bottom of the active site crevasse by four endogenous ligands and a solvent molecule (Wat827). The orientations of the endogenous ligands are relatively unperturbed in each inhibitor complex, but the inhibitors themselves bind to or near the iron in a range of positions, all of which perturb the position of Wat827. The three lowest-affinity inhibitors (MHP, PHP, and IHB) yield distorted trigonal bipyramidal iron coordination geometry in which the inhibitor C4-phenolate group displaces the solvent ligand. MHB binds within the active site, but neither its C3-OH group nor the solvent molecule binds to the iron. The C4-phenolate group of the three highest-affinity inhibitors (PHB, CHB, and FHB) coordinates the Fe3+ adjacent to Wat827, resulting in a shift in its position to yield a six-coordinate distorted octahedral geometry. The range of inhibitor orientations may mimic the mechanistically significant stages of substrate binding to 3, 4-PCD. The structure of the final substrate complex is reported in the following paper [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066].
Orville
A M
AM
Department of Biochemistry, Medical School, and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455-0347, USA.
Elango
N
N
Lipscomb
J D
JD
Ohlendorf
D H
DH
D001665:Binding Sites; D001667:Binding, Competitive; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011520:Protocatechuate-3,4-Dioxygenase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016958:Pseudomonas putida / Q000201:enzymology
D004791:Enzyme Inhibitors; D008024:Ligands; D011520:Protocatechuate-3,4-Dioxygenase