Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase

Journal Article · Tue Feb 06 00:00:00 EST 2018 · Plant Physiology (Bethesda)

DOI:https://doi.org/10.1104/pp.17.01608· OSTI ID:1410860

Jun, Se-Young ^[1]; Sattler, Steven A.  ^[2]; Cortez, Gabriel S. ^[1]; Vermerris, Wilfred  ^[3]; Sattler, Scott E.  ^[4]; Kang, ChulHee  ^[5]

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1. Department of Chemistry, Washington State University, Pullman, Washington 99164
2. School of Molecular Biosciences, Washington State University, Pullman, Washington 99163
3. Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, and Genetics Institute, University of Florida, Gainesville, Florida 32610
4. United States Department of Agriculture-Agricultural Research Service, Wheat, Sorghum, and Forage Research Unit, and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68583
5. Department of Chemistry, Washington State University, Pullman, Washington 99164, School of Molecular Biosciences, Washington State University, Pullman, Washington 99163

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.

• Research Organization: Univ. of Florida, Gainesville, FL (United States)
• Sponsoring Organization: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office; USDOE Office of International Affairs (IA); National Science Foundation (NSF); National Inst. of Health; Biomass Research and Development Initiative; AFRI; CRIS
• Grant/Contract Number: PI0000031; DBI 0959778; 1R01GM11125401; 2011-1006-30358; 2011-67009-30026; 3042-21220-032-00D
• OSTI ID: 1410860
• Alternate ID(s): OSTI ID: 1661473; OSTI ID: 1766557
• Journal Information: Plant Physiology (Bethesda), Journal Name: Plant Physiology (Bethesda) Vol. 176 Journal Issue: 2; ISSN 0032-0889
• Publisher: American Society of Plant Biologists
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES