An active site homology model of phenylalanine ammonia‐lyase from P. crispum

Abstract

The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in k cat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615-and 14-fold lower k cat , respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.

Isolation and characterization of the anthocyanidin genes pal, f3h and dfr of Scutellaria viscidula (Lamiaceae)

“…Some highly conserved residues were found in the , Q 497 and 3, 5-dihydro-5-methylidene-4H-imidazol-4-one (MIO), which were considered to be of significant importance for substrate binding, catalytic activity and LMIO formation (Xu et al, 2008). The mechanism was in agreement with the recent report from Petroselinum crispum research (Röther et al, 2002). In particular, SvPAL contained a conserved region named Ala-Ser-Gly triad, which can be converted autocatalytically, via internal cyclization and elimination of two water molecules, to the electrophilic prosthetic group MIO (Song and Wang, 2009) (Figure 5).…”

Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase

“…The active center residues of PAL were also confirmed by mutational studies guided by a homology model based on HAL (Rö ther et al, 2002). Most intriguingly, the mutation Tyr110/Phe resulted in a complete loss of activity, although this Tyr should not be that important for the reaction as it is expected to contact merely the substrate carboxylate group.…”

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

“…This approach identified at least two putative cyanobacterial PAL enzymes that were each annotated initially as HALs. The A. variabilis and N. punctiforme proteins were predicted to have PAL activity as they contained an aliphatic residue conserved in the active site of known PAL enzymes thought to be associated with substrate specificity (Supplementary data) (28). These two cyanobacterial PALs are 77% identical in amino-acid sequence and have significant sequence similarity to plant PAL enzymes, although they are more similar in size (AvPAL is 567 residues, NpPAL is 569 residues) to the smaller prokaryotic HAL enzymes (P. putida HAL is 509 residues) than the larger plant PAL enzymes (P. crispum PAL is 715 residues).…”

“…In previous studies of ammonia-lyases, a number of active-site residues were proposed to be responsible for conferring substrate selectivity for L-His or L-Phe (1,28). One residue of interest, His 83 (P. putida numbering) in HAL is thought to orient L-His in the active site, whereas the substitution of more hydrophobic residues at the corresponding position in PAL proteins may serve to better accommodate the substrate L-Phe (28).…”