Publications

• Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis.

  Calabrese J.C., Jordan D.B., Boodhoo A., Sariaslani S., Vannelli T.

  The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically related histidine ammonia lyase (HAL), with PAL having an additional approximately 16 ... >> More

  The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically related histidine ammonia lyase (HAL), with PAL having an additional approximately 160 residues extending from the common fold. We propose that catalysis (including lowering the pK(a) of nonacidic C3 of l-phenylalanine for an E1cb mechanism) is potentially governed by dipole moments of seven alpha helices associated with the PAL active site (six positive poles and one negative pole). Cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) resides atop the positive poles of three helices, for increasing its electrophilicity. The helix dipoles appear fully compatible with a model of phenylalanine docked in the active site of PAL having the first covalent bond formed between the amino group of substrate and the methylidene group of MIO: 12 highly conserved residues (near the N termini of helices for enhancing function) are poised to serve roles in substrate recognition, MIO activation, product separation, proton donation, or polarizing electrons from the phenyl ring of substrate for activation of C3; and a highly conserved His residue (near the C terminus of the one helix that directs its negative pole toward the active site to increase the residue's basicity) is positioned to act as a general base, abstracting the pro-S hydrogen from C3 of substrate. A similar mechanism is proposed for HAL, which has a similar disposition of seven alpha helices and similar active-site residues. The helix dipoles appear incompatible with a proposed mechanism that invokes a carbocation intermediate. << Less

  Biochemistry 43:11403-11416(2004) [PubMed] [EuropePMC]

• The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms.

  Cochrane F.C., Davin L.B., Lewis N.G.

  In Arabidopsis thaliana, four genes have been annotated as provisionally encoding PAL. In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be catalytically competent for l-phenylalanine deamination, whereas AtPAL3, obtained as a N-terminal His-tagged protein, was of very low ac ... >> More

  In Arabidopsis thaliana, four genes have been annotated as provisionally encoding PAL. In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be catalytically competent for l-phenylalanine deamination, whereas AtPAL3, obtained as a N-terminal His-tagged protein, was of very low activity and only detectable at high substrate concentrations. All four PALs displayed similar pH optima, but not temperature optima; AtPAL3 had a lower temperature optimum than the other three isoforms. AtPAL1, 2 and 4 had similar K(m) values (64-71 microM) for l-Phe, with AtPAL2 apparently being slightly more catalytically efficacious due to decreased K(m) and higher k(cat) values, relative to the others. As anticipated, PAL activities with l-tyrosine were either low (AtPAL1, 2, and 4) or undetectable (AtPAL3), thereby establishing that l-Phe is the true physiological substrate. This detailed knowledge of the kinetic and functional properties of the various PAL isoforms now provides the necessary biochemical foundation required for the systematic investigation and dissection of the organization of the PAL metabolic network/gene circuitry involved in numerous aspects of phenylpropanoid metabolism in A. thaliana spanning various cell types, tissues and organs. << Less

  Phytochemistry 65:1557-1564(2004) [PubMed] [EuropePMC]

• A modern view of phenylalanine ammonia lyase.

  MacDonald M.J., D'Cunha G.B.

  Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microo ... >> More

  Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed. << Less

  Biochem Cell Biol 85:273-282(2007) [PubMed] [EuropePMC]

• Structural investigations into the stereochemistry and activity of a phenylalanine-2,3-aminomutase from Taxus chinensis.

  Wybenga G.G., Szymanski W., Wu B., Feringa B.L., Janssen D.B., Dijkstra B.W.

  Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-α-phenylalanine into (R)-β-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic ... >> More

  Phenylalanine-2,3-aminomutase (PAM) from Taxus chinensis, a 4-methylidene-imidazole-5-one (MIO)-dependent enzyme, catalyzes the reversible conversion of (S)-α-phenylalanine into (R)-β-phenylalanine via trans-cinnamic acid. The enzyme also catalyzes the direct addition of ammonia to trans-cinnamic acid, a reaction that can be used for the preparation of β-amino acids, which occur as frequent constituents of bioactive compounds. Different hypotheses have been formulated to explain the stereochemistry of the PAM-catalyzed reaction, but structural evidence for these hypotheses is lacking. Furthermore, it remains unclear how the PAM MIO group is formed from the three-amino acid (A-S-G) sequence motif. For these reasons, we elucidated PAM three-dimensional (3D) structures with a bound (R)-β-phenylalanine analogue and with bound trans-cinnamic acid. In addition, 3D structures of the (inactive) Y322A and N231A mutants of PAM were elucidated, which were found to be MIO-less. We conclude that the stereochemistry of the PAM-catalyzed reaction originates from the enzyme's ability to bind trans-cinnamic acid in two different orientations, with either the si,si face or the re,re face directed toward the MIO group, as evidenced by two distinct carboxylate binding modes. The results also suggest that the N231 side chain promotes MIO group formation by increasing the nucleophilicity of the G177 N atom through acidification of the amide proton. << Less

  Biochemistry 53:3187-3198(2014) [PubMed] [EuropePMC]

• Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonia-lyases.

  Louie G.V., Bowman M.E., Moffitt M.C., Baiga T.J., Moore B.S., Noel J.P.

  Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-c ... >> More

  Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for beta-amino acid biosynthesis. << Less

  Chem. Biol. 13:1327-1338(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.