title: Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.
abstract: Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Torrens-Spence
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Lazear
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States.
authors.forename: Renee
authors.initials: R
authors.lastname: von Guggenberg
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States.
authors.forename: Haizhen
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States. Electronic address: lij@vt.edu.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics; D020134:Catalytic Domain; D030008:Catharanthus / Q000201:enzymology* / Q000235:genetics; D005998:Glycine / Q000235:genetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010207:Papaver / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity; D014445:Tyrosine Decarboxylase / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D012694:Serine; D014445:Tyrosine Decarboxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases; D005998:Glycine
keywords: Apocynaceae; Aromatic amino acid decarboxylase; Catharanthus roseus; Papaver somniferum; Papaveraceae; Tryptophan decarboxylases; Tyrosine decarboxylases