Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from L-phenylalanine to give trans-cinnamate. In monocots, PAL also displays tyrosine ammonia lyase activity (TAL), leading to the formation of p-coumaric acid. The catalytic mechanism and substrate-specificity of a major PAL from Sorghum bicolor (SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared to that of dicotyledonous plants. The sidechain of His123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from 189Ala-Ser-Gly191 residues and the bound L-phenylalanine and L-tyrosine, conferring the deamination reaction through either Friedel-Crafts or E2 reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. This enhancement could establish the basis for further engineering sorghum PALs for potential benefits on both silage/forage quality as well as production of renewable fuels and chemicals from plant biomass. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate-preference among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality. {copyright, serif} 2017 American Society of Plant Biologists. All rights reserved.

Citation

Se-Young Jun, Steven A Sattler, Gabriel S Cortez, Wilfred Vermerris, Scott E Sattler, ChulHee Kang. Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase. Plant physiology. 2017 Dec 01

PMID: 29196539

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