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A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

Ronau JA, Paul LN, Fuchs JE, Liedl KR, Abu-Omar MM, Das C - Biochemistry (2014)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Purdue University , 560 Oval Drive, West Lafayette, Indiana 47907, United States.

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.

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Electron density (colored green) for active site metal-coordinatingresidues, cobalt (M), and mutant side chains of D139 (colored yellow)for (a) D139E, (b) D139N, (c) D139A, and (d) D139K. The maps shownare Fo – Fc simulated annealing omit maps contoured at 3σ.
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fig7: Electron density (colored green) for active site metal-coordinatingresidues, cobalt (M), and mutant side chains of D139 (colored yellow)for (a) D139E, (b) D139N, (c) D139A, and (d) D139K. The maps shownare Fo – Fc simulated annealing omit maps contoured at 3σ.

Mentions: As discussed earlier, the pterin cofactoris known to bind to cPAH through several side chain contacts, oneof which is mediated through three interactions of water with asparticacid 139. Because the overall crystal structures for the mutants werealmost identical to that of the wild-type enzyme, we focused our analysison the pterin binding pocket of the active site. A hydrogen bond networkconsisting of waters 1–10 (Figure 5a,PDB entry 3TK4)31 bridges the active site metal withthe carboxylate group of Asp139, the carbonyl group of Pro117, thehydroxyl group of Tyr179, and the carboxylate of E184 (which alsocoordinates the metal in a bidentate fashion). Because of improvedresolution, water molecules constituting this network have been assignedon the basis of the 1.5 Å resolution crystal structure of cPAHbound to Co31 instead of the 2.0 Åstructure of it bound to Fe (PDB entry 1LTV).15 Bindingof pterin in the active site (PDB entry 1LTZ) induces a displacement of waters 6 and7, due to a steric clash with the O4 atom of the pterin moiety. Waters9 and 10 are also lost to accommodate the dihydroxypropyl chain ofpterin, while a new water molecule is seen hydrogen bonding with bothwaters 1 and 8 (Figure 5b,c). Since we wereunable to obtain structures of the mutants in the pterin-bound state,we focused our analysis on the solvation state of the mutants in theirmetalated form, prior to binding of the cofactor (Figure 6). The B factors for waters contributingto the hydrogen bonding network in the pterin pocket are listed inTable S1 of the Supporting Information.Electron density for active site residues, mutated side chains forD139, and the active site metal are shown in an Fo – Fc simulated annealingomit map contoured at 3σ (Figure 7).


A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

Ronau JA, Paul LN, Fuchs JE, Liedl KR, Abu-Omar MM, Das C - Biochemistry (2014)

Electron density (colored green) for active site metal-coordinatingresidues, cobalt (M), and mutant side chains of D139 (colored yellow)for (a) D139E, (b) D139N, (c) D139A, and (d) D139K. The maps shownare Fo – Fc simulated annealing omit maps contoured at 3σ.
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fig7: Electron density (colored green) for active site metal-coordinatingresidues, cobalt (M), and mutant side chains of D139 (colored yellow)for (a) D139E, (b) D139N, (c) D139A, and (d) D139K. The maps shownare Fo – Fc simulated annealing omit maps contoured at 3σ.
Mentions: As discussed earlier, the pterin cofactoris known to bind to cPAH through several side chain contacts, oneof which is mediated through three interactions of water with asparticacid 139. Because the overall crystal structures for the mutants werealmost identical to that of the wild-type enzyme, we focused our analysison the pterin binding pocket of the active site. A hydrogen bond networkconsisting of waters 1–10 (Figure 5a,PDB entry 3TK4)31 bridges the active site metal withthe carboxylate group of Asp139, the carbonyl group of Pro117, thehydroxyl group of Tyr179, and the carboxylate of E184 (which alsocoordinates the metal in a bidentate fashion). Because of improvedresolution, water molecules constituting this network have been assignedon the basis of the 1.5 Å resolution crystal structure of cPAHbound to Co31 instead of the 2.0 Åstructure of it bound to Fe (PDB entry 1LTV).15 Bindingof pterin in the active site (PDB entry 1LTZ) induces a displacement of waters 6 and7, due to a steric clash with the O4 atom of the pterin moiety. Waters9 and 10 are also lost to accommodate the dihydroxypropyl chain ofpterin, while a new water molecule is seen hydrogen bonding with bothwaters 1 and 8 (Figure 5b,c). Since we wereunable to obtain structures of the mutants in the pterin-bound state,we focused our analysis on the solvation state of the mutants in theirmetalated form, prior to binding of the cofactor (Figure 6). The B factors for waters contributingto the hydrogen bonding network in the pterin pocket are listed inTable S1 of the Supporting Information.Electron density for active site residues, mutated side chains forD139, and the active site metal are shown in an Fo – Fc simulated annealingomit map contoured at 3σ (Figure 7).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Purdue University , 560 Oval Drive, West Lafayette, Indiana 47907, United States.

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.

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