|Calabrese, Joseph - DUPONT CTRL RES DEV|
|Boodhoo, Amechand - DUPONT CTRL RES DEV|
|Sariaslani, Sima - DUPONT CTRL RES DEV|
|Vannelli, Todd - DUPONT CTRL RES DEV|
Submitted to: Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 8, 2004
Publication Date: September 14, 2004
Citation: Calabrese, J.C., Jordan, D.B., Boodhoo, A., Sariaslani, S., Vannelli, T. 2004. Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Journal of Biochemistry. 43:11403-11416. Interpretive Summary: The enzyme, phenylalanine ammonia lyase, mediates the first steps of biosynthetic pathways that produce lignins, coumarins, and more rare natural products. There is interest in improving its activity so that it can be used efficiently in industrial applications. In order to make the improvements, it is highly useful to understand how the enzyme works. We propose a mechanism of enzyme action that is based on the three-dimensional structure of the protein, which we determined at atomic resolution. The proposal provides a starting point for engineering improved enzyme that is industrially useful.
Technical Abstract: The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 angstrom resolution using PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically-related histidine ammonia lyase (HAL), with PAL having an additional ~160 residues extending from the common fold. We propose that catalysis (including lowering the pKa 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 towards 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.