Abstract
Benzoic acid decarboxylases offer an elegant alternative to CO2 fixation by reverse reaction-carboxylation, which is named the bio-Kolbe–Schmitt reaction, but they are unfavorable to carboxylation. Enhancing the carboxylation efficiency of reversible benzoic acid decarboxylases is restricted by the unexplained carboxylation mechanisms. The direction of reversible enzyme catalytic reactions depends on whether catalytic residues at the active center of the enzyme are protonated, which is subjected by the pH. Therefore, the forward and reverse reactions could be separated at different pH values. Reversible 2,3-dihydroxybenzoate acid decarboxylase undergoes decarboxylation at pH 5.0 and carboxylation at pH 8.6. However, it is unknown whether the interaction of enzymes with substrates and products in the forward and reverse reactions can be exploited to improve the catalytic activity of reversible enzymes in the unfavorable direction. Here, we identify a V-shaped tunnel of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) through which the substrate travels in the enzyme, and demonstrate that the side chain conformation of a tyrosine residue controls the entry and exit of substrate/product during reversible reactions. Together with the kinetic studies of the mutants, it is clarified that interactions between substrate/product traveling through the enzyme tunnel in 2,3-DHBD_Ao are direction-dependent. These results enrich the understanding of the interactions of substrates/products with macromolecular reversible enzymes in different reaction directions, thereby demonstrating a possible path for engineering decarboxylases with higher carboxylation efficiency.
Key points
• The residue Trp23 of 2,3-DHBD_Ao served as a switch to control the entry and exit of catechol
• A V-shaped tunnel of 2,3-DHBD_Ao for decarboxylation and carboxylation reactions was identified
• The results provide a promising strategy for engineering decarboxylases with direction-dependent residues inside the substrate/product traveling tunnel of the enzyme
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Acknowledgements
We thank Shijun Zhong from School of Bioengineering, Dalian University of Technology, for his comment and suggestion on the mechanism analysis. We cordially thank the staff of beamline BL17U1 and BL18U1 at the Shanghai Synchrotron Radiation Facility, People’s Republic of China, for assistance in synchrotron X-ray data collection.
Funding
This study was financially supported by the National Natural Science Foundation of China (No. 21877110) and National Key Research and Development Program (No. 2021YFC2103702).
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Y. F. designed and conducted most of the experiments, analyzed the results, and wrote the manuscript. S. W. conducted the theoretical calculations. Y. F. and X. S. analyzed the data and wrote the manuscript. J. S. and X. L. provided the useful suggestions for experiments. All authors read and approved the final manuscript.
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Fan, Y., Wu, S., Shi, J. et al. The catalytic mechanism of direction-dependent interactions for 2,3-dihydroxybenzoate decarboxylase. Appl Microbiol Biotechnol 107, 7451–7462 (2023). https://doi.org/10.1007/s00253-023-12813-9
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DOI: https://doi.org/10.1007/s00253-023-12813-9