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

Jun, Se-Young; Sattler, Steven A.; Cortez, Gabriel S.; Vermerris, Wilfred; Sattler, Scott E.; Kang, ChulHee

doi:10.1104/pp.17.01608

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

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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 canmore »« less

Authors:

Jun, Se-Young ^[1];

^[2]; Cortez, Gabriel S. ^[1];

^[3];

^[4];

^[5]

Department of Chemistry, Washington State University, Pullman, Washington 99164
School of Molecular Biosciences, Washington State University, Pullman, Washington 99163
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, and Genetics Institute, University of Florida, Gainesville, Florida 32610
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
Department of Chemistry, Washington State University, Pullman, Washington 99164, School of Molecular Biosciences, Washington State University, Pullman, Washington 99163

Publication Date:: Tue Feb 06 00:00:00 EST 2018

Research Org.:: Univ. of Florida, Gainesville, FL (United States)

Sponsoring Org.:: 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

OSTI Identifier:: 1410860

Alternate Identifier(s):: OSTI ID: 1661473; OSTI ID: 1766557

Grant/Contract Number:: PI0000031; DBI 0959778; 1R01GM11125401; 2011-1006-30358; 2011-67009-30026; 3042-21220-032-00D

Resource Type:: Published Article

Journal Name:: Plant Physiology (Bethesda)

Additional Journal Information:: Journal Name: Plant Physiology (Bethesda) Journal Volume: 176 Journal Issue: 2; Journal ID: ISSN 0032-0889

Publisher:: American Society of Plant Biologists

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES

Citation Formats


                    Jun, Se-Young, Sattler, Steven A., Cortez, Gabriel S., Vermerris, Wilfred, Sattler, Scott E., and Kang, ChulHee. Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase.  United States: N. p., 2018. 
Web.  doi:10.1104/pp.17.01608.

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                    Jun, Se-Young, Sattler, Steven A., Cortez, Gabriel S., Vermerris, Wilfred, Sattler, Scott E., & Kang, ChulHee. Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase.  United States.  https://doi.org/10.1104/pp.17.01608

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                    Jun, Se-Young, Sattler, Steven A., Cortez, Gabriel S., Vermerris, Wilfred, Sattler, Scott E., and Kang, ChulHee. Tue .  
"Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase".  United States.  https://doi.org/10.1104/pp.17.01608.

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@article{osti_1410860,

  title        = {Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase},

  author       = {Jun, Se-Young and Sattler, Steven A. and Cortez, Gabriel S. and Vermerris, Wilfred and Sattler, Scott E. and Kang, ChulHee},

  abstractNote = {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.},

  doi          = {10.1104/pp.17.01608},

  journal      = {Plant Physiology (Bethesda)},

  number       = 2,

  volume       = 176,

  place        = {United States},

  year         = {Tue Feb 06 00:00:00 EST 2018},

  month        = {Tue Feb 06 00:00:00 EST 2018}

}

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