Crystal Structure of Phenylalanine Ammonia Lyase:  Multiple Helix Dipoles Implicated in Catalysis^,

Abstract

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.

Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix espanaensis

“…A conserved Ala-Ser-Gly segment which is typical for ammonia-lyases can also be found in sam8 (4,24). Furthermore, several active-site amino acid residues proposed for HAL after elucidation of the X-ray structure of HutH, a HAL from Pseudomonas putida, have analogous residues in Sam8 (22,24).…”

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

“…It is also the only general phenylpropanoid pathway enzyme for which detailed structure-function information is available. The first structures of bacterial and plant PALs were solved in 2004 [10,55]. Other bacterial and fungal PAL/TAL structures were elucidated more recently [45,47].…”

Discovery of Two Cyanobacterial Phenylalanine Ammonia Lyases: Kinetic and Structural Characterization,

“…In TAL, the first of these segments is packed tightly within the active-site cleft and interacts closely with the bound substrate/product molecule, whereas the second segment forms a more external loop that caps the active-site region. These loops appear to be intrinsically flexible in the PAL proteins, as they are also poorly ordered in the structures of parsley and yeast PAL (9,10). In the crystal structures of the cyanobacterial PALs, the active-site cleft appears very exposed to the external solvent, and the absence of stabilizing interactions contributed by the disordered inner loop is a likely reason for the failure to observe stable binding to these PALs of the substrate (L-Phe), the product (cinnamic acid), and a potent PAL-specific inhibitor (2-aminoindan-2-phosphonic acid) (31).…”

“…In total, these structure determinations showed that the ammonia-lyases exist as homotetramers possessing a conserved polypeptide-chain fold. The eukaryotic PALs are ~20 kDa per monomer larger than the prokaryotic HALs, by virtue of a 54-residue N-terminal extension and an inserted 122-residue domain (9)(10)(11). The additional domain in the plant and fungal enzymes forms an arch over the active site and has been proposed to function as a shielding domain by restricting substrate entry and product egress (10).…”