Crystallographic and mutational evaluation of substrate binding in phenylalanine hydroxylase from Chromobacterium violaceum

Judith Adele Ronau, Purdue University

Abstract

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. The binding with phenylalanine at this site has been confirmed by isothermal titration calorimetry (ITC), revealing a dissociation constant of 24 ± 1.1 μM, yet no detectable binding was seen under the same conditions with alanine and tyrosine. Point mutations in the distal site (F258A, Y155A, T254A) lead to impaired binding as probed by ITC, with the mutants exhibiting a discernible defect in their catalytic activity. However, x-ray structures of Y155A and F258A show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PAH is a member of the aromatic amino acid hydroxylase (AAAH) family; each member catalyzes the hydroxylation of their aromatic amino acid substrate. The catalytic domains of AAAHs are all similar and the active sites contain a non heme iron atom coordinated to a 2-His-1-carboxylate facial triad and two waters. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and hydrogen bonds with one of these water molecules and to two additional water molecules that do not ligate iron. All three waters are observed crystallographically forming hydrogen bonds with Asp139 and an oxidized form of the cofactor, 7,8-dihydro-L-biopterin (BH2). To determine the catalytic role of this residue, point mutants D139N, D139A, D139E, and D139K were prepared and characterized. Investigation of the mutants reveals this residue plays an important role in both iron and pterin binding. The binding of iron in the active site requires the presence of a polar residue of similar size to D139 at this position, as the affinity of D139N for iron is the same as wild type cPAH, while the affinity of D139A for iron is markedly decreased. Strikingly, polarity is not the sole criterion for metal affinity, as size of the amino acid side chain also appears to be important. Despite most AAAHs containing Glu at this position instead of Asp as in cPAH, substitution of Asp for Glu also results in decreased iron affinity. While the presence of a polar residue of similar size to Asp in this position is sufficient for iron binding, the negative charge of the carboxylate group of Asp is crucial for proper binding of the cofactor. D139N, which binds iron similarly as wild type cPAH has a reduced affinity for pterin as measured by isothermal titration calorimetry. Furthermore, its catalytic efficiency for phenylalanine and pterin are 17-fold and 25-fold decreased respectively. These findings are consistent with Asp139 having an important role in binding of iron, maintaining proper binding of pterin through indirect interactions with the cofactor, therefore playing an essential role in catalytic efficiency of the enzyme.

Degree

Ph.D.

Advisors

Das, Purdue University.

Subject Area

Biochemistry|Biophysics

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