A Conserved Acidic Residue in Phenylalanine HydroxylaseContributes to Cofactor Affinity and Catalysis
Thecatalytic domains of aromatic amino acid hydroxylases (AAAHs)contain a non-heme iron coordinated to a 2-His-1-carboxylate facialtriad and two water molecules. Asp139 from Chromobacteriumviolaceum PAH (cPAH) resides within the second coordinationsphere and contributes key hydrogen bonds with three active site watersthat mediate its interaction with an oxidized form of the cofactor,7,8-dihydro-l-biopterin, in crystal structures. To determinethe catalytic role of this residue, various point mutants were preparedand characterized. Our isothermal titration calorimetry (ITC) analysisof iron binding implies that polarity at position 139 is not the solecriterion for metal affinity, as binding studies with D139E suggestthat the size of the amino acid side chain also appears to be important.High-resolution crystal structures of the mutants reveal that Asp139may not be essential for holding the bridging water molecules together,because many of these waters are retained even in the Ala mutant.However, interactions via the bridging waters contribute to cofactorbinding at the active site, interactions for which charge of the residueis important, as the D139N mutant shows a 5-fold decrease in its affinityfor pterin as revealed by ITC (compared to a 16-fold loss of affinityin the case of the Ala mutant). The Asn and Ala mutants show a muchmore pronounced defect in their kcat values,with nearly 16- and 100-fold changes relative to that of the wildtype, respectively, indicating a substantial role of this residuein stabilization of the transition state by aligning the cofactorin a productive orientation, most likely through direct binding withthe cofactor, supported by data from molecular dynamics simulationsof the complexes. Our results indicate that the intervening waterstructure between the cofactor and the acidic residue masks directinteraction between the two, possibly to prevent uncoupled hydroxylationof the cofactor before the arrival of phenylalanine. It thus appearsthat the second-coordination sphere Asp residue in cPAH, and, by extrapolation,the equivalent residue in other AAAHs, plays a role in fine-tuningpterin affinity in the ground state via deformable interactions withbridging waters and assumes a more significant role in the transitionstate by aligning the cofactor through direct hydrogen bonding.
[…] The program
molrep from the ccp4 suite was used to determine the structure using a
structure of apo-cPAH (PDB entry 1LTU). For molrep,
all water molecules and other ions such as iron and chloride were
removed from the aforementioned model to limit model bias. Several
cycles of refinement and model building proceeded using Refmac5 and Coot, respectively.
Anisotropic B factors were used during the refinement
of all D139 mutant structures. Weights were also optimized during
the refinement process. The crystallographic data and refinement statistics
are listed in Table . All figures were rendered
with PYMOL (version 220.127.116.11). […]