Computational protocol: Conserved Molecular Mechanism of TyrA Dehydrogenase Substrate Specificity Underlying Alternative Tyrosine Biosynthetic Pathways in Plants and Microbes

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Protocol publication

[…] BlastP searches were performed using the amino acid sequences of previously characterized TyrA homologs from plants [soybean PDH; GmPDH1 (Schenck et al., ) and Arabidopsis ADH; AtADH2 (Rippert and Matringe, )] and microbes [Synechocystis sp. PCC6803 ADH (Legrand et al., ), and E. coli PDH (Hudson et al., )] as the query in the NCBI database. This yielded only closely-related plant and microbial TyrA orthologs (e.g., algae and, γ-proteobacteria), which were then used as the query to perform additional BlastP searches. Every 5th BlastP hit was selected to provide sequences from various microbial lineages and limit bias in sample selection. Data contains all the sequence information for the TyrA orthologs used in Figure and Figure . A structure-guided amino acid alignment was performed in PROMALS3D (Pei and Grishin, ) using the default parameters with structures of TyrA enzymes from plants and microbes with varying substrate specificities (G. max TyrAp; GmPDH1; PDB # 5T8X, H. influenzae TyrAp; HiPDH; 2PV7, and Synechocystis sp. PCC6803 TyrAa; SynADH; PDB # 2F1K). The amino acid alignment from PROMALS3D was used to construct phylogenetic trees using MEGA7 (Kumar et al., ). The full amino acid alignment can be found in Data . The analyses involved 130 amino acid sequences and all sites with <75% coverage were eliminated from the analysis. A neighbor-joining method (Figure ; Saitou and Nei, ) was used to estimate evolutionary history using 1,000 bootstrap replicates (values shown at branches). The tree in Figure is a representative tree using a subset of the sequences found in Figure . Additional phylogenetic analyses were performed using the Maximum Likelihood method based on the Jones-Taylor-Thornton (JTT) matrix-based model (Jones et al., ), which gave overall similar results (Figure ). All phylogenetic trees are drawn to scale, with branch lengths measured in the number of substitutions per site. [...] Homology models were made using SWISS-MODEL (Biasini et al., ) with default parameters to predict the structures of divergent TyrA enzymes. Enzymes that are more closely-related to plants (e.g., SsTyrA and MhTyrA) were modeled using the GmPDH1 structure as the template, though this resulted in a poor model for BdTyrA, which falls within the outgroup. BdTyrA was additionally modeled using Synechocystis sp. PCC6803 ADH. Homology models were visualized using PyMOL. […]

Pipeline specifications

Software tools SWISS-MODEL, PyMOL
Application Protein structure analysis
Organisms Homo sapiens
Chemicals Dopamine, Epinephrine, Morphine, Tyrosine, Vitamin E