Univ. of Alabama, Birmingham, AL (United States). Dept. of Chemistry and Biochemistry
Univ. of Texas, Arlington, TX (United States). Dept. of Chemistry and Biochemistry
Case Western Reserve Univ., Cleveland, OH (United States). Dept. of Pharmacology
Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II)
Univ. of Washington, Seattle, WA (United States). Depts. of Biological Structure and Biochemistry
Univ. of California, Irvine, CA (United States). School of Medicine, Dept. of Ophthalmology
Univ. of California, Irvine, CA (United States). School of Medicine, Dept. of Ophthalmology; Univ. of California, Irvine, CA (United States). School of Medicine, Dept. of Physiology and Biophysics; VA Long Beach Healthcare System, CA (United States). Research Service
Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme–substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.
York, Nicholas J., et al. "Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center." Journal of Biological Chemistry, vol. 296, Dec. 2020. https://doi.org/10.1016/j.jbc.2021.100492
York, Nicholas J., Lockart, Molly M., Sardar, Sinjinee, Khadka, Nimesh, Shi, Wuxian, Stenkamp, Ronald E., Zhang, Jianye, Kiser, Philip D., & Pierce, Brad S. (2020). Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center. Journal of Biological Chemistry, 296. https://doi.org/10.1016/j.jbc.2021.100492
York, Nicholas J., Lockart, Molly M., Sardar, Sinjinee, et al., "Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center," Journal of Biological Chemistry 296 (2020), https://doi.org/10.1016/j.jbc.2021.100492
@article{osti_1778798,
author = {York, Nicholas J. and Lockart, Molly M. and Sardar, Sinjinee and Khadka, Nimesh and Shi, Wuxian and Stenkamp, Ronald E. and Zhang, Jianye and Kiser, Philip D. and Pierce, Brad S.},
title = {Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center},
annote = {Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme–substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.},
doi = {10.1016/j.jbc.2021.100492},
url = {https://www.osti.gov/biblio/1778798},
journal = {Journal of Biological Chemistry},
issn = {ISSN 0021-9258},
volume = {296},
place = {United States},
publisher = {American Society for Biochemistry and Molecular Biology},
year = {2020},
month = {12}}