NSRRC Activity Report 2022

050 NSRRC ACTIVITY REPORT 2022 (TIM barrel) fold that consists of a central β-barrel surrounded by several helices ( Fig. 1(a) ). The protein harbors three large flexible loops (loop A, B, and C) on the top of the barrel, which are located close to the putative substrate entry site and are presumably related to substrate selectivity ( Fig. 1(a) ). Glyphosate was seen to be located adjacent to the nicotinamide moiety of the NADP + in a cavity that is formed by residues Trp21, Tyr49, Leu287, His111, Trp112, and Phe291 ( Fig. 1(b) ). The carboxylate group of glyphosate formed H-bond networks with Tyr49, His111, Trp21, and the O7N of NADP + . The residues Leu287, Trp112, and Phe291 may provide hydrophobic interactions with glyphosate. Because the glyphosate observed in the structure is located near the catalytic tetrad and nicotinamide ring of NADP + where hydride transfer can take place, the cavity enclosing the glyphosate is presumed to be the authentic substrate- binding site. In addition, a variant F291D of AKR4C17 that was constructed based on structure-based engineering exhibited a 70% increase in glyphosate degradation ( Fig. 1(c) ). AKRs are widely distributed in prokaryotes and eukaryotes and can be robustly classified into 16 subfamilies on the basis of sequence identity. 5 Members of families 1, 6, and 7 are found in humans and other mammals, whereas the majority of plant AKRs belongs to AKR4. 5 AKR4 includes A, B, and C subfamilies, with A playing a role in plant-microbe interactions, B in iron acquisition from the soil, and C in stress defense. 6,7 The research team also determined that residues that were responsible for glyphosate- binding were considerably conserved in AKR4C1–7 and 9–15. 4 Thus, these AKR members are expected to have the potential to degrade glyphosate as well as AKR4C17. This study provides molecular insights into the mechanism of action of AKR-mediated glyphosate degradation and greatly contribute to the deployment and application of glyphosate (Reported by Rey-Ting Guo, Hubei University, China). This report features the work of Rey-Ting Guo and his collaborators published in J. Hazard. Mater. 436 , 129191 (2022). TPS 05A Protein Microcrystallography TLS 15A1 Biopharmaceuticals Protein Crystallography • Protein Crystallography • Biological Macromolecules, Protein Structures, Life Science References 1. E. Schönbrunn, S. Eschenburg, W. A. Shuttleworth, J. V. Schloss, N. Amrhein, J. N. S. Evans, W. Kabsch, PNAS 98 , (a) (b) (c) Fig. 1 : Structural and functional investigation of the glyphosate-binding residues of AKR4C17. (a) The overall folding of AKR4C17, the α-helix, β-sheet, and loop region are colored red, yellow, and green, respectively. (b) The interaction network of AKR4C17 and glyphosate. The 2 F o – F c (grey mesh) and F o – F c omit (blue mesh) map of glyphosate were contoured at 1.0 and 3.0 σ, respectively. Glyphosate, NADP + , catalytic- tetrad residues, and glyphosate-interacting residues are colored yellow, pink, blue, and green, respectively. The distances are labeled in the unit of Å. (c) The glyphosate-degrading activity of each mutant is presented as a percentage of wild-type AKR4C17. Each measurement was performed in triplicate. Error bars that indicate standard deviations are presented for each group. ND = not detectable. [Reproduced from Ref. 4] 1376 (2001). 2. T. A. Gaines, E. L. Patterson, P. Neve, New Phytol. 223 , 1770 (2019). 3. L. Pan, Q. Yu, H. Han, L. Mao, A. Nyporko, L. Fan, L. Bai, S. Powles, J. Plant Physiol. 181 , 1519 (2020). 4. H. Li, Y. Yang, Y. Hu, C. C. Chen, J. W. Huang, J. Min, L. Dai, R. T. Guo, J. Hazard. Mater. 436 , 129191 (2022). 5. T. M. Penning, Chem. Biol. Interact. 234 , 236 (2015). 6. J. M. Jez, M. J. Bennett, B. P. Schlegel, M. Lewis, T. M. Pen- ning, Biochem. J. 326 , 625 (1997). 7. D. Sengupta, D. Naik, A. R. Reddy, J. Plant Physiol. 179 , 40 (2015).