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Insights into the Binding Interaction of Catechol 1,2-Dioxygenase with Catechol in Achromobacter xylosoxidans DN002

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Abstract

Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme–substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme.

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Data Availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Funding

The authors would like to acknowledge the support received from the National Natural Science Foundation of China (31000069), and the Key Project on Social Development of Science and Technology in Shaanxi Province (grant no. 2017ZDXM-SF-105).

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  1. Shaanxi Provincial Key Laboratory of Biotechnology, Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, 229 Taibai North Rd, Xi’an, Shaanxi, 710069, People’s Republic of China

    Yani Liu, Fengdan Wei, Rui Xu, Tao Cheng & Yanling Ma

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  1. Yani Liu
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Contributions

YL, FW, and YM conceived and designed the study. YL, RX, and TC performed the experiments and bioinformatics analysis. YL, FW, and RX analyzed the data and prepared all of figures. YL, RX, and YM wrote the manuscript. YL and YM revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yanling Ma.

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Liu, Y., Wei, F., Xu, R. et al. Insights into the Binding Interaction of Catechol 1,2-Dioxygenase with Catechol in Achromobacter xylosoxidans DN002. Appl Biochem Biotechnol 195, 298–313 (2023). https://doi.org/10.1007/s12010-022-04129-7

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