Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation
Graphical abstract
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously present in the environment and have harmful effects on humans [1]. However, some PAHs, such as 1-naphthol, are essential ingredients for polymers, dyes, herbicides, insecticides and pharmaceutical preparations [[2], [3], [4]]. Hence there is an interest in useful transformations of PAHs, such as producing 1-naphthol from naphthalene. However, current methods of manufacturing naphthols require acids, bases, and metal catalysts [3], none of which is environmentally friendly, cheap, or disposable.
Biocatalysis offers the advantage of performing reactions under mild conditions with minimal requirements and provides a more environmentally benign approach than conventional industrial methods [5,6]. Oxygenases are a class of enzymes that have great potential and versatility for catalyzing reactions that are generally not accessible by chemical routes with high regio-, stereo-, and enantioselectivities [3,[7], [8], [9]]. To date, most studies of the biocatalytic synthesis of 1-naphthol have focused on monooxygenases. For example, Canada et al. [3] reported a mutant of toluene ortho-monooxygenase, which is capable of synthesizing 1-naphthol [[10], [11], [12]]. Despite this enzyme possessing a high potential for industrial applications, it is a three-component enzyme, so engineering improvements to the protein is unfortunately complicated. Other efforts include those by Molina-Espeja et al. [13], who engineered an extracellular fungal aromatic peroxygenase with monooxygenase activity that used hydrogen peroxide and was able to convert naphthalene into 1-naphthol. However, 2-naphthol was always present as a byproduct in their study.
Cytochrome P450 monooxygenase is another prominent oxygenase that produces 1-naphthol. There are many reports on evolved P450BM3 variants that oxidize the non-natural substrate naphthalene [[14], [15], [16]]. CYP505D6 is also reported to hydroxylate naphthalene and naphthols [17]. P450s are among the most widely studied oxygenases and can be made to catalyse numerous promiscuous and even non-natural reactions [18]. P450 s are found in all kingdoms and domains of life and concern dozens of reaction types and compounds [18]. The investigation of P450 modification strategies not only will facilitated the production of 1-naphthol, but also has potential applications in the fields of pharmacy, pesticides, and material
P450 monooxygenase CYP116B3 from Rhodococcus ruber strain DSM 44319, is reported to have oxidation activity towards polycyclic aromatic hydrocarbons including naphthalene. Naphthalene can be oxidized regioselectivity to 1-naphthol by CYP116B3 [18]. However, the natural substrate of CYP116B3 is 7-ethoxycoumarin, and although naphthalene oxidation can be catalyzed by CYP116B3, it is limited by low conversion efficiency. The key to improving catalytic efficiency may be changes in the substrate recognition sites (SRSs), which are the regions in the enzyme involved in recognition and binding of substrates [19]. The function of SRS1, SRS2 and SRS3 is to recruit and accommodate the substrates, while SRS4, SRS5 and SRS6 stabilize the protein structure [20]. The evolutionary rate of SRS1-3 is significantly faster than that of SRS4-5, so SRS1-3 mutagenesis is usually the focus of investigation [20]. Therefore, the directed evolution in the SRS1-3 position in CYP116B3 might be significant for improving 1-naphthol production and providing a deeper understanding of CYP116B3.
In this work, a homology model of CYP116B3 was used to identify putative SRS amino acid residues and determine the tunnel for naphthalene diffusion from the protein surface. We then employed directed evolution to tailor CYP116B3 for naphthalene hydroxylation. Our strategy employed site-directed and saturation mutagenesis, and beneficial mutations were combined to create the most-active variant. The strategy demonstrated here could provide an alternative platform to enhance the catalytic activity of CYP116B3 in hydroxylation reactions and improve the PAH production.
Section snippets
Chemicals, bacterial strains and plasmids
Naphthalene, 1-naphthol, and 2-naphthol were purchased from Shanghai Lingfeng Chemical Reagent Co. (Shanghai, China). The 7-ethoxycoumarin and 7-hydroxycoumarin were obtained from Energy Chemical Co. (Shanghai, China). All other chemicals were of analytical grades. Bacterial strains and pET28a are described in “Whole-cell reaction” below.
Homology modelling and docking simulations
The heme domain structure of CYP116B3 was obtained by homology modelling using Modeller 9.11 [21]. P450terp, CYP105A1, CYP130 and P450 Pikc (PDB: 1CPT, 2ZBX,
Identification of target residues with substrate recognition sites
At present, there is no report on the structure of CYP116B3. Four proteins with 30–40 % sequence identity with CYP116B3 were used as templates for multi-template homology modelling. However, there were almost no identical consecutive sequence between CYP116B3 and the template. The putative structure of the heme domain in CYP116B3 was obtained. In most P450 s, SRS1 lies in the highly variable loop region between αB and αC (BC-loop), and SRS2 is located in the C-terminal end of αF, while SRS3
Discussion
Recent efforts have been made to improve phenolics production, including with the use of enzymes [24,25]. However, since a single microbe is unlikely to have the enzymes necessary for efficient PAH hydroxylation, enzyme modification is an important step for the biological production of phenolic compounds. This study presents a strategy for making modifications to P450 monooxygenase to achieve high levels of 1-naphthol production in a single-phase E. coli system.
The mutant CMABC had 3 acid amino
Conclusions
For the improvement in the hydroxylation activity of CYP116B family enzymes, it is worth noting that selection of mutational target residues should be done in the SRS1-3, especially residues at the entrance of the substrate access tunnel and in the active site. In this research, some amino acid residues have been found to increase the yield of specific products. Although single mutations are not necessarily cooperative, especially for those in vicinal sites, the CYP116B3 E88C/N199Q/Q209A mutant
Formatting of funding sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Sha Tao: Conceptualization, Software, Writing - original draft. Yang Gao: Formal analysis, Investigation. Kang Li: Visualization, Validation. Qiuhao Lu: Resources, Software. Chenggang Qiu: Methodology, Conceptualization. Xin Wang: Supervision, Visualization. Kequan Chen: Writing - review & editing, Supervision. Pingkai Ouyang: Supervision.
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
This work has been supported by the National Key Research and Development Program of China (Grant No. 2016YFA0204300), Key Research and Development Program (Social Development) Project of Jiangsu Province (BE2018730).
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