Premium
Structural basis of distinct salicylic acid glucosylation in Arabidopsis thaliana by two homologous enzymes: implications for plant stress response
Author(s) -
Thompson Alayna Michelle George,
Iancu Cristina V,
Dean John,
Choe Junyong
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1142.3
Subject(s) - salicylic acid , arabidopsis thaliana , biochemistry , enzyme , in silico , chemistry , arabidopsis , homology modeling , amino acid , docking (animal) , transferase , gene , mutant , medicine , nursing
The objective of this work is to understand SA‐glucose conjugate formation by two enzymes from Arabidopsis thaliana, UGT74F1 and UGT74F2. Salicylic acid (SA) is utilized as a signaling molecule by plants in response to various stresses. To store SA in an inactive form, conjugates are formed with small organic molecules such as glucose, and inactive SA can then be transported and stored in plant vacuoles. In the model organism Arabidopsis thaliana SA‐glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP‐glucose to SA. Despite being 77% identical with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms both SAG and salicylic acid glucose ester (SGE). SAG and SGE serve different cellular functions in terms of storage, so understanding their creation is important. As UGT74F1 and UGT74F1 are very similar, there are likely very subtle differences responsible for the observed final products. To investigate the difference between these enzymes, we began with crystallizing purified proteins. We solved the crystal structure of UGT74F2 complexed with its substrates UDP and SA, and this structure allowed the identification the residues that coordinate SA – H18, Y180, and M274. These residues are conserved between UGT74F1 and UGT74F2, but the loops containing these residues have several amino acid substitutions. In silico docking of SA to UGT74F1 (homology model) and UGT74F2 suggests that SA binding could be more constrained in UGT74F1. From our crystal structure and in silico modeling, we theorize that amino acid substitutions around the binding site are responsible for the observed activity differences between these enzymes. Some substitutions could affect hydrogen bonding: position 15 is threonine in UGT74F2, but serine in UGT74F1, while position 112 is alanine in UGT74F2 but a serine in UGT74F1. Other substitutions could drastically change the geometry of loops near the binding pocket, such as position 178 which is either a leucine (UGT74F1) or a proline (UGT74F2). For our future directions, we are investigating the involvement of these substitutions in product formation. We have produced mutant proteins and are investigating their products by enzymatic assays. Additionally, we are using in silico ligand docking, x‐ray scattering on solution phase protein, and x‐ray diffraction crystallography to investigate the subtle differences between UGT74F1 and UGT74F2. Support or Funding Information Supported by RFUMS‐DePaul Pilot Grant Award