A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

Abstract

The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may 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 cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of phenylalanine. It thus appears that the second-coordination sphere Asp residue in cPAH, and, by extrapolation, the equivalent residue in other AAAHs, plays a role in fine-tuning pterin affinity in the ground state via deformable interactions with bridging waters and assumes a more significant role in the transition state by aligning the cofactor through direct hydrogen bonding.

Pterin function in bacteria

“…The exact chemical identity of this pterin cofactor is flexible, as MPt, dimethyl pterin, and BPt have all been reported to be sufficient for proper enzymatic activity [24,96,97]. The pterin cofactor is coordinated in the enzymatic active site by a network of hydrogen bonds and a conserved aspartate residue on the cognate protein [98]. This aspartate residue is hypothesized to be essential for maintaining proper orientation of the cofactor to support productive catalysis.…”

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579

“…Phenylalanine hydroxylases (PAHs) are enzymes that catalyze the hydroxylation of phenylalanine to tyrosine (Fig. 1; Kappock & Caradonna, 1996;Erlandsen et al, 2002;Fitzpatrick, 2003;Ronau et al, 2013Ronau et al, , 2014. These enzymes belong to the aromatic amino-acid hydroxylase (AAAH) family, which includes tyrosine hydroxylases (THs) and tryptophan hydroxylases (TPHs) (Hufton et al, 1995;Fitzpatrick, 2003;Cao et al, 2010;Ronau et al, 2014;Skjaerven et al, 2014).…”

“…1; Kappock & Caradonna, 1996;Erlandsen et al, 2002;Fitzpatrick, 2003;Ronau et al, 2013Ronau et al, , 2014. These enzymes belong to the aromatic amino-acid hydroxylase (AAAH) family, which includes tyrosine hydroxylases (THs) and tryptophan hydroxylases (TPHs) (Hufton et al, 1995;Fitzpatrick, 2003;Cao et al, 2010;Ronau et al, 2014;Skjaerven et al, 2014). AAAHs are nonheme iron(II)-containing enzymes and utilize (6R)-5,6,7,8-tetrahydrobiopterin (BH 4 ) as a cofactor (Cao et al, 2010;Skjaerven et al, 2014).…”

“…AAAHs are nonheme iron(II)-containing enzymes and utilize (6R)-5,6,7,8-tetrahydrobiopterin (BH 4 ) as a cofactor (Cao et al, 2010;Skjaerven et al, 2014). The BH 4 cofactor is converted to 7,8-dihydrobiopterin (BH 2 ) during the enzymatic reaction of the corresponding AAAH and is then recycled to BH 4 by pterin 4a-carbiolamine dehydratase and dihydropteridine reductase (Crabtree & Channon, 2011;Ronau et al, 2014).…”

Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction

“…Moreover, the residues related to enzymatic activity and substrate binding in CvPAH were identified using molecular modification. Tyr179 plays a role in substrate binding and the ratedetermining step in catalytic processes [13], and Asp139 contributes to stabilization of the transition state by direct binding with the cofactor [14]. CvPAH displayed low Ltryptophan hydroxylation activity, and the double variant L101Y/W180F exhibited a 5.2-fold increase in activity compared to the wild type [15].…”