Glyphosate, an organophosphorus compound, is the most extensively employed herbicide in agriculture worldwide including in Thailand, due to its efficient weed elimination. The widespread of glyphosate in water and soil is caused by inappropriate application practices and over-spraying herbicide, thus affecting the potential risks to human health. Moreover, the detection of glyphosate residues in human urine samples results in an increased exposure to glyphosate in humans. Currently, the selective analysis of glyphosate in environmental samples provides an early warning in incidents of contamination, thus being important for public health and food security.

           This research work investigated the capability of the aldo-keto reductases (AKRs) in glyphosate degradation, thus improving glyphosate potency in weed management. AKR Superfamily comprises enzymes that have broad substrate specificity and play specific roles in reactive aldehyde and ketone detoxification. In this study, AKRs, designated as AKR4C16 and AKR4C17, were found to contribute to glyphosate resistance. 

           The genes (EcAKR4–1 and EcAKR4–2) designated as AKR4C16 and AKR4C17, respectively in an AKR Superfamily demonstrated glyphosate degradation, thus suggesting the potential role in glyphosate detoxification. The control rice plants were killed by applied glyphosate at rates of 540 g ha-1 or higher as shown in Figure 1. However, the EcAKR4-1-overexpressing rice died at 2,160 g glyphosate ha-1 but it still survived at 540 and 1,080 glyphosate g ha-1 (Figure 1).

            Growth response to glyphosate was compared between the GFP (control) and EcAKR4-1 gene (Figure 1), therefore AKR4C16 confers glyphosate resistance in rice. In conclusion, the enzyme activity for glyphosate degradation was significantly higher in the transgenic lines expressing AKR4C16 compared to the wild type.

Figure 1 The GFP (control) and transgenic rice seedlings after three weeks of glyphosate treatment. 

             

              Crystal structures of AKR4C16 and AKR4C17 have been studied by the Macromolecular Crystallography technique. Refined structures of AKR4C16 and AKR4C17 adopt the same overall fold of a triosephosphate isomerase (TIM) motif. Their cofactor- and putative substrate-binding pockets are primarily identical. Therefore, AKR4C16 and AKR4C17 might exploit the same enzymatic mechanism to catalytically degrade glyphosate.

              The catalytic process of glyphosate biodegradation is proposedly illustrated in Figure 2. The ternary complex structure reveals that both its cofactor and glyphosate are located at the long tunnel of the AKR4C17 structure as shown in Figure 2. The binding regions of NADP+ and glyphosate are displayed in green and yellow on the protein surface of AKR4C17, respectively.

Figure 2 The putative catalytic process and the cofactor and substrate-binding site of AKR4C17.

            Thereafter, the F291D mutant obtained from structure-based rational design enhances a 70% increase in enzyme activity for glyphosate degradation. Proposedly, the mutant structure preserved the overall fold and cofactor-binding features of wild-type enzymes. More hydrophilic interactions may assist glyphosate biodegradation, thus altering the electrostatic surface of the putative substrate-binding site.

            The dimensional structure of AKR4C17, an aldo-keto reductase could investigate its role in the glyphosate detoxification process, thus increasing the degree of glyphosate tolerance. Therefore, information obtained including the function and molecular structure of AKR4C147 might be very important in developing glyphosate-tolerant plants in the future.

Re-written by Chomphunuch Songsiriritthigul (Beamline Scientist, SLRI)

References

1. Li, H., Yang, Y., Hu, Y., Chen, C-C., Huang, J-W., Min, J., Dai, L., Guo, R-T. (2022). Structural analysis and engineering of aldo-keto reductase from glyphosate-resistant Echinochloa colona. Journal of Hazardous Materials. DOI: 10.1016/j.jhazmat.2022.129191.
2. Butmee, P., Tumcharern, G., Songsiriritthigul, C., Durand, M., J., Thouand, G., Kerr, M., Kalcher, K. & Samphao, A. (2021). Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition. Analytical and Bioanalytical Chemistry. DOI: 10.1007/s00216-021-03567-2.
3. Songsiriritthigul, C., Narawongsanont, R., Tantitadapitak, C., Guan, H-H. & Chen, C-J. (2020). Structure-function study of AKR4C14, an aldo-keto reductase from Thai Jasmine rice (Oryza sativa L. ssp. Indica cv. KDML105. Acta Crystallographica D. D76, 472-483.
4. Pan, L., Yu, Q., Han, H., Mao, L., Nyporko, A., Fan, L., Bai, L., Powles, S. (2019). Aldo-keto reductase metabolizes glyphosate and confers glyphosate resistance in Echinochloa colona. Plant Physiology. 181 (4), 1519–1534. DOI: 10.1104/pp.19.00979.