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

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 -cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (; 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 Ala-Ser-Gly residues and the bound l-phenylalanine and l-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E 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.

Phenylalanine and Tyrosine as Exogenous Precursors of Wheat (Triticum aestivum L.) Secondary Metabolism through PAL-Associated Pathways

“…PTAL can use both Tyr and Phe with equal efficiency. It has been noted before that PTAL in monocotyledons has demonstrated PAL/TAL activity ratio between 0.2 and 6 [19,[32][33][34].…”

Large scale genome-wide association study reveals that drought induced lodging in grain sorghum is associated with plant height and traits linked to carbon remobilisation

“…Twelve of the 21 genes involved in lignin biosynthesis (Bout and Vermerris 2003; Saballos et al 2009, 2012, Sattler et al 2009, 2017, Walker et al 2013, 2016, Jun et al 2017, 2018; Wang et al 2017) were located within genomic regions associated with lodging in this study (Fig. 2), representing a significant enrichment (χ 2 test, p value = 0.008).…”

“…Twelve of the 21 genes involved in lignin biosynthesis (Bout and Vermerris, 2003; Jun et al , 2017, 2018, Saballos et al , 2012, 2009, Sattler et al , 2009, 2017, Walker et al , 2013, 2016; Wang et al , 2017) were located within genomic regions associated with lodging in this study (Figure 2), representing a significant enrichment (χ 2 test, p value = 0.008). Lodging resistant sorghum generally have more lignification, both in the number of cells with lignified walls and in the degree of lignification in the epidermis, sub-epidermis, and vascular bundles (Schertz and Rosenow, 1977).…”

“…Brown vertical bars to the right of each chromosome marked as “LDG” represent the locations of QTL previously detected for lodging (Kebede et al , 2001; Murray et al , 2008; Srinivasa Reddy et al , 2008); the black bars indicate the location of a QTL previously reported for charcoal rot and/or Fusarium stalk rot (Srinivasa Reddy et al 2008; Adeyanju et al 2015). The location of genes in the lignin biosynthesis pathways that co-located with lodging QTL detected in this study are highlighted in green to the right of each chromosome, and those that did not co-locate with lodging QTL are highlighted in blue (Bout and Vermerris 2003; Saballos et al 2009, 2012, Sattler et al 2009, 2017, Walker et al 2013, 2016, Jun et al 2017, 2018; Wang et al 2017). The location of the recently cloned Dry gene is highlighted in red (Zhang et al 2018).…”

“…A limited number of genes for stem composition have been identified and functionally validated in sorghum. Based on phylogenetic relationships with functionally confirmed genes in sorghum and other species, predicted translational protein functions, enzymatic activity assay, and gene expression patterns, 21 candidate genes in the lignin biosynthesis pathways have been identified in sorghum (Bout and Vermerris 2003; Saballos et al 2009, 2012, Sattler et al 2009, 2017, Walker et al 2013, 2016, Jun et al 2017, 2018; Wang et al 2017). Additionally, the major morphological D gene, which changes the structure of the sorghum pith, was recently cloned (Zhang et al 2018).…”