Reversible photocontrol of oxidase activity by inserting a photosensitive domain into the oxidase
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Photocontrol of protein activity has become a helpful strategy for regulating biological pathways. Herein, a method for the precise and reversible photocontrol of oxidase activity was developed by using the conformational change of the AsLOV2 domain.
The AsLOV2 domain was inserted into the nonconserved sites exposed on the surface of the AdhP protein, and the alov9 fusion was successfully screened for subsequent optical experiments under the assumption that neither of these actions affected the original activity of AdhP protein. The activity of alov9 was noticeably inhibited when the fusion was exposed to 470 nm blue light and recovered within 30 min. As a result, we could precisely and reversibly photocontrol alov9 activity through the optimization of several parameters, including cofactor concentration, light intensity, and illumination time.
KeywordsAsLOV2 Enzyme activity Insertion Oxidase Photocontrol
LOV2 domain isolated from Avena sativa
optical density of bacteria at 600 nm
Photocontrol of protein dynamics is a powerful tool for the precise spatial and temporal control of signal transduction (Pathak et al. 2014; Zhang and Cui 2015). The light-sensitive LOV2 domain of Avena sativa phototropin 1 (AsLOV2) is part of the PAS superfamily of domains (Crosson and Moffat 2001), each of which consists of a flavin nucleotide and a C-terminal Jα helix (Harper et al. 2003; Halavaty and Moffat 2007). During the conformational change of the AsLOV2 domain, the Jα helix will unfold under illumination (Harper et al. 2004; Swartz et al. 2002), and thus it has been utilized to construct engineered optical switches. As a photosensitive element, the AsLOV2 domain has many advantages, including high spatial and temporal resolution, fast response to blue light, and good reversibility (Lee et al. 2014). Therefore, the AsLOV2 domain has been widely used as an engineered light switch in recent years.
There are two general approaches to achieve the photocontrol of proteins based on the AsLOV2 domain. One is based on the peptide-tagged LOV2 domain (Kawano et al. 2015). Devin et al. developed tunable light-inducible dimerization tags (TULIPs) to control protein localization (Strickland et al. 2012). The other approach is based on peptide-binding (Lungu et al. 2012). Hui et al. constructed the LOVTRAP system for reversible light-induced protein dissociation (Wang et al. 2016). Most studies have focused on interfering protein–protein interactions (PPIs) using the photosensitive domain, but studies have only rarely concentrated on the kinetics of individual proteins. Here, a method was introduced for achieving reversible photocontrol of oxidase AdhP in time and space based on insertion of the AsLOV2 domain.
In our work, the LOV2 domain was inserted into surface-exposed and evolutionarily nonconserved sites of the AdhP protein to prevent possible structural perturbations. The AdhP was cloned from E. coli MG1655 and catalyzes the oxidation–reduction reactions of short-chain alcohols and aldehydes (Thomas et al. 2013). The 10 Å spacing between its N and C termini prevented structural interference of the original protein (Dagliyan et al. 2016). Therefore, we assumed that when the LOV2 domain underwent a conformational change upon illumination, its disorder can be efficiently transmitted to the active center of the AdhP protein to produce a structural disturbance, which would inhibit the activity of the AdhP protein.
Materials and methods
Cloning, expression, and purification of enzymes
The gene for AdhP was subcloned into pET-28a(+) via BamHI/XhoI (Thermo Fisher Scientific, Rockford, IL, USA) restriction sites to create an in-frame N-terminal 6*histidine tag. The LOV2 and adhP genes were spliced together using the splicing by overlap extension polymerase chain reaction method (SOE-PCR) (the primer information is listed in Additional file 1: Table S1) (Chen et al. 2016). The plasmids were then transformed into Escherichia coli BL21 (DE3)-competent cells.
The recombinant E. coli BL21 (DE3) was incubated in lysogeny broth (LB) containing 50 mg/L kanamycin at 37 °C. When the OD600 reached approximately 0.6, enzyme expression was induced by the addition of 0.1 mM IPTG (final concentration). The culture was incubated at 18 °C for an additional 12 h at 200 rpm. After centrifugation (6000×g, 10 min, 4 °C) and washing with 0.9% NaCl, the cells were resuspended in 20 mM sodium phosphate buffer (pH 8.5) and disrupted by ultrasonication. After centrifugation (12,000×g, 30 min, 4 °C), the crude extract was used for protein purification using a 5-mL HisTrap FF crude column (GE Healthcare, Waukesha, WI, USA). The protein was eluted with an increasing gradient from 20 to 500 mM of imidazole in sodium phosphate buffer. Proteins were evaluated using SDS-PAGE, and the concentration was conducted using a Bradford assay kit (Beyotime, Shanghai, China). The purified enzyme was concentrated and stored at − 20 °C for further use.
Enzyme activity was determined by measuring the rate of NAD+ reduction at 340 nm on an ultraviolet (UV)/visible spectrophotometer (BioTek Instruments, Winooski, VT, US); a molar extinction coefficient of 6.22 mM−1 cm−1 for NADH was used for the calculation (Zhang et al. 2002). Standard reactions were performed in 200 μL of sodium phosphate buffer (100 mM, pH 8.5) at 30 °C containing 0.2 mM NAD+, 5 mM substrate (in 5% v/v DMSO), and an appropriate amount of purified enzyme. One unit of enzyme activity was defined as the reduction of 1 μmol geraniol per minute. The light group activity was measured immediately after irradiation for a precise period. Specific wavelength 470 nm LED light sources was used for blue light illumination. All of the measurements were carried out over 2 min and performed at least three times.
Structure analysis and homology modeling
The structural model of alov9 was built using the modeler 9.20 program (Schwede et al. 2003). The crystal structure of AdhP (PDB ID: 4kgv) and AsLOV2 domain (PDB ID: 5hzj) were used as templates. Model evaluation was performed using the Ramachandran plot Web server (Betteridge et al. 2003). Ramachandran plot analysis results showed that 95.4% of the total number of residues was in the favored and allowed region for alov9. The model could be considered reliable only if the percentage of residues in the favored and allowed region was greater than 90%. Thus, the evaluation results indicated that our model of alov9 was reliable. Structural analysis was performed using the PyMOL 1.8.0 program.
Results and discussion
Strategies to inhibit AdhP activity
To establish whether fusion enzyme activity remains stable after insertion into the sensitive domain, activity assays were performed in a catalytic system using a microplate reader. The results showed that the activity of these 10 fusions had varying degrees of reduction except the insertion of ninth point (Thr215) (Fig. 1c), which might be caused by the insertion of AsLOV2 at the ninth site on the surface of the AdhP protein and prevents influencing the correct folding of the AdhP protein. The results confirmed that insertion of LOV2 domain causes different structural interferences to the AdhP protein. From the perspective of maintaining stable protein activity, alov9 fusion was selected for a subsequent study.
Optical inhibition enzyme activity
The FMN cofactor is necessary for the unfolding of the AsLOV2 protein in the blue state (Christie et al. 1998). It has been confirmed that the incorporation of cofactor FMN is required for the light sensitivity of alov9 (Yu et al. 2016). As expected, the catalytic activity of alov9 in the absence of cofactor FMN did not decrease under irradiation (Fig. 2b). The data indicated that the participation of cofactor FMN is required in the next optical experiment.
Exploration of factors affecting optical suppression
Reversible regulation of alov9
In summary, based on the conformational change of the light-sensitive AsLOV2 domain, a method was successfully developed to reversibly photocontrol oxidase activity. We successfully constructed the alov9 fusion, in which the AsLOV2 domain was inserted into the allosteric sites of the AdhP protein, without interfering in the original AdhP activity. This alov9 fusion was indeed strongly inhibited upon exposure to blue light. Our results showed that alov9 fusion activity can be reversibly photocontrolled and still retain 87% of its initial activity after four illumination cycles. Finally, oxidase activity can achieve spatial and temporal control in a precise time. The control of protein activity via LOV2 insertion may prove to be a valuable tool for other biological processes in the future.
This work was funded by the National Natural Science Foundation of China (No. 21778018), Natural Science Foundation of Shanghai (19ZR1412700), and Research Program of State Key Laboratory of Bioreactor Engineering.
TS conducted the experiments. TS and BZ drafted the manuscript. YR provided advice in the experiments design and data analysis. All authors read and approved the final manuscript.
This work was funded by the National Natural Science Foundation of China (No. 21778018), Natural Science Foundation of Shanghai (19ZR1412700) and Research Program of State Key Laboratory of Bioreactor Engineering.
Ethics approval and consent to participate
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Consent for publication
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The authors declare that they have no competing interests.
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