Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

Zoidakis, Jérôme; Loaiza, Aristobulo; Vu, Kim; Abu‐Omar, Mahdi M.

doi:10.1016/j.jinorgbio.2004.12.017

Cited by 21 publications

(18 citation statements)

References 17 publications

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“…One hundred percent activity in the PAH-1 and the PAH-2 reactions occurred at 37°C under the aforementioned conditions. The optimal temperatures for PAH-1 and PAH-2 are in accordance with data previously obtained for humans and Dictyostelium discoideum (58,59) but are different from data obtained for Chromobacterium violaceum, Colwellia psychrerythraea, and Chloroflexus aurantiacus (13,60,61). PAH-1 activity decreased quickly at higher temperatures and was limited at 50°C (Fig.…”

Section: Resultssupporting

confidence: 81%

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Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Wang

Chen

Hao

et al. 2013

Appl Environ Microbiol

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c Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4␣-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalaninehydroxylating system is functionally significant in lipid metabolism.

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Section: Resultssupporting

confidence: 81%

“…4B), which is similar to the optimum pH for recombinant human and C. violaceum PAHs (59,60). Maximal activity for both PAH-1 and PAH-2 occurred at pH 7.0 under the aforementioned conditions.…”

Section: Resultssupporting

confidence: 68%

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Wang

Chen

Hao

et al. 2013

Appl Environ Microbiol

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“…3A). The activation energy of the reaction catalyzed by CpPAH (E a ϭ 4.9 Ϯ 0.3 kcal/mol) is lower than for mesophilic CvPAH (13), indicating that CpPAH possesses cold-adapted features. Studies of psychrophilic enzymes show that they have lowered their E a in order to reduce the temperature dependence of k cat , making catalysis less affected by a decrease in temperature (49,50).…”

Section: Resultsmentioning

confidence: 89%

Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability

Leiros

Pey

Innselset

et al. 2007

Journal of Biological Chemistry

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The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). . Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

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“…One possible explanation for this is that the lysine mutant has greater affinity for an inhibitory metal such as cobalt (used in the crystallization buffer) rather than iron. 57 Alternatively, the metal-bound species of the Lys mutant could be a minor population in solution, but one that has crystallized preferentially over the major, nonmetalated species. The kinetic and binding results with the lysine mutant may reflect a steric clash with the cofactor, despite our observation in crystals that it could be oriented toward the metal center.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2014

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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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Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum

Cited by 21 publications

References 17 publications

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability

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

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