Inorganic Chemistry
Article
group of TXP formed a hydrogen bond (H-bond) with the
distal Asp64, thus stabilizing the binding of TXP to the protein.
The substrate was further stabilized by the H-bond with Lys67.
Moreover, the binding was enhanced by the π−π stacking
between the benzene rings of TXP and Phe49. As shown in
stronger binding compared to that of TCP, which agrees with
the lower Km value for TBP binding to A15C/H64D Ngb
(Table 1). These observations may because TBP has a large
van der Waals force compared to that of TCP binding to the
protein. In addition, the complex structure showed that the
binding of TXP affects the H-bond network around the heme-
coordinated water molecule and the catalytic Asp64-Lys67
pair, which provides structural information for the TXP-
induced UV−Vis spectral change at 630 nm (Figures 2A and
over native DHP in catalytic efficiency but also eliminates the
substrate inhibition effect caused by the internal binding
model.53 It should be noted that although the catalytic
efficiency cannot match that of our previously designed
artificial dehaloperoxidase, F43Y/H64D Mb,39 the kcat of
A15C/H64D Ngb was increased by 60% without an inhibition
effect of TXP. Because the wastewater could be contaminated
by both halophenols and industrial dyes, the study suggests
that A15C/H64D Ngb is an efficient multifunctional
peroxidase, which has potential applications in bioremediation
for environmental sustainability.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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sı
In the case of RB19 binding, it showed that the dye bound
to the heme edge on the protein surface where a series of
interactions occurred (Figures 6B and 7A). The terminal
Experimental details, UV−Vis spectra, mass spectra,
kinetic studies, and docking results (PDF)
AUTHOR INFORMATION
Corresponding Authors
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Lianzhi Li − School of Chemistry and Chemical Engineering,
Liaocheng University, Liaocheng 252059, China;
Ying-Wu Lin − School of Chemistry and Chemical
Engineering, University of South China, Hengyang 421001,
China; Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
Authors
Shun-Fa Chen − School of Chemistry and Chemical
Engineering, University of South China, Hengyang 421001,
China
Figure 7. Binding of RB19 to A15C/H64D Ngb. (A) 2D interaction
diagram between RB19 and protein in the docking model with the
lowest energy. (B) ITC data for titration of 10 μM A15C/H64D Ngb
with 500 μM RB19 in 100 mM potassium phosphate buffer (pH 7.0)
at 25 °C. The top panel shows the raw data, and the bottom panel
shows the integrated heat versus substrate/enzyme ratio. The data are
fitted to a one-site binding model.
Xi-Chun Liu − School of Chemistry and Chemical Engineering,
University of South China, Hengyang 421001, China
Jia-Kun Xu − Key Lab of Sustainable Development of Polar
Fisheries, Ministry of Agriculture and Rural Affairs, Lab for
Marine Drugs and Byproducts of Pilot National Lab for
Marine Science and Technology, Yellow Sea Fisheries
Research Institute, Chinese Academy of Fishery Sciences,
Qingdao 266071, China
Jia-Jia Lang − Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
421001, China
Ge-Bo Wen − Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
421001, China
sulfate group of RB19 formed multiple H-bonds with Asn45
and Lys67 near the distal heme site, and the 2-sulfate group on
the anthracene ring formed an H-bond with Lys95. Major van
der Waals interactions include those between the anthracene
ring and Leu70, the benzene ring, and the side chain of Lys67.
These protein−substrate contacts resulted in a moderate
binding affinity of RB19 to A15C/H64D Ngb. This was further
supported by the isothermal titration calorimetry (ITC) study,
which showed a one-site binding model, with an association
constant of 1.32 0.04 × 104 M−1 (Figure 7B).
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CONCLUSIONS
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Author Contributions
In summary, we have successfully converted human Ngb into
an efficient peroxidase, A15C/H64D Ngb, with both
dehalogenation and dye-decolorizing activities even exceeding
some of native enzymes. The rationally designed acidic Asp64
together with native Lys67 at the heme distal site enhanced the
activation rate of H2O2, presumably through the acid−base
catalytic mechanism.41,52 The Asp64 substitution also facili-
tated the substrate binding as compared to the Ser64
substitution. A15C/H64D Ngb not only has an advantage
S.-F.X., X.-C.L., and J.-K.X. contributed equally. The manu-
script was written through contributions of all authors. All
authors have given approval to the final version of the
manuscript.
Funding
This work was supported by the National Natural Science
Foundation of China (no. 21977042), Special Project of Major
Scientific and Technological Innovation in Shandong Province
2843
Inorg. Chem. 2021, 60, 2839−2845