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ARS Home » Plains Area » Lincoln, Nebraska » Wheat, Sorghum and Forage Research » Research » Publications at this Location » Publication #342732
Title: Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase

Author
item JUN, SE-YOUNG - Washington State University
item VERMERRIS, WILFRED - University Of Florida
item Sattler, Scott
item KANG, CHULHEE - Washington State University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2017
Publication Date: 2/1/2018
Publication URL: https://handle.nal.usda.gov/10113/6477311
Citation: Jun, S., Vermerris, W., Sattler, S.E., Kang, C. 2018. Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia-lyase. Plant Physiology. 174:1452-1468. https://dol.org/10.1104/pp.17.01608.
DOI: https://doi.org/10.1104/pp.17.01608

Interpretive Summary: In the U.S., sorghum biomass (stalks and leaves) serves as an important forage crop for livestock. In addition, sorghum is being developed as a bioenergy crop for cellulosic biofuels. Cellulosic biofuels are derived from the breakdown the cell wall polymers (cellulose and hemicellulose) of the biomass to sugars and the conversions of these sugars to fuel molecules. A third cell wall polymer, lignin, makes cell walls resistant to breakdown either in livestock digestive systems or in the cellulosic conversion process. Phenylalanine Ammonia-Lyase (PAL) is an enzyme, which is involved in the first step needed to make the subunits of lignin. In this study, the structure of this enzyme was determined in order to understand how PAL functions in chemical reactions. Changing key features of this enzyme changed the products of these chemical reactions. This study opens a new door toward tailoring lignin levels in plants to improve conversion biomass into biofuels or forage utilization of livestock.

Technical Abstract: 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 (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (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 with that of dicotyledonous plants. The side chain of histidine-123 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 the 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. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences 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.