Elsevier

Journal of Catalysis

Volume 384, April 2020, Pages 30-36
Journal of Catalysis

Electrochemically boosted cytochrome P450 reaction that efficiently produces 25-hydroxyvitamin D3

https://doi.org/10.1016/j.jcat.2020.02.012Get rights and content

Highlights

  • Valuable 25-hydroxyvitamin D3 was produced by coupling P450 enzyme and electrode.

  • Electrons were efficiently supplied to P450 through mediators.

  • Undesired electron loss and enzyme deactivation were significantly reduced.

  • The present system worked with the crude P450 sample without purification.

Abstract

We investigated electrochemical P450 vitamin D3 hydroxylase (Vdh) reactions using promiscuous spinach ferredoxin and a small organic molecule as the mediators that shuttle electrons from the electrode to the P450. The choice of small mediator and modified electrode surface were crucially important because they decrease electron loss to undesired reactions and reduce the amount of enzyme-deactivating reactive oxygen species produced. Twice the amount of 25-hydroxyvitamin D3 was produced under optimum conditions compared to that obtained by the conventional in vitro conversion system using NADH as the electron source, which demonstrates the electrochemical boost provided to the P450 reaction. We also confirmed that the present system works with crude extracts from Escherichia coli containing Vdh, which shows that it is possibly useful for a wide variety of P450 applications.

Introduction

Ubiquitous cytochrome P450 enzymes (P450s) form a tremendously diverse superfamily of heme-containing bio-catalysts that are functionally versatile [1], [2], [3]. Given their wide variety of reactions, P450s are promising tools in the pharmaceutical and chemical industries [4], [5], [6], [7], [8], [9], [10]. 25-Hydroxyvitamin D3 (25(OH)VD3) is an important pharmaceutical compound for the treatment of a variety of diseases, such as rickets, osteoporosis, and psoriasis, among others [11]. However, the selective hydroxylation of C-H bonds is a challenging process in synthetic chemistry, hence, the utilization of P450 is advantageous. It has been reported that an actinobacterium (Pseudonocardia autotrophica) produces 25(OH)VD3 [12], [13], and it possesses a total of 28 P450 genes, one of which encodes VD3 25-hydroxylase (Vdh) [14]. Vdh is a typical prokaryotic water-soluble P450, and a large amount of Vdh can be obtained using bacterial recombinant expression systems [14], [15], [16].

A typical P450 reaction requires the sequential transfer of two electrons from the reduced form of nicotinamide adenine dinucleotide (phosphate), NAD(P)H, to the heme iron in the enzyme, which is considered to be the rate-limiting step [17]. This process is species-dependent and complicated by the involvement of one or two redox partner proteins as natural electron mediator(s), making it difficult to construct systems that apply P450 reactions in vitro. The natural redox partner of the aforementioned Vdh is yet unexplored, and an efficient system for the in vitro production of 25(OH)VD3 has not yet been achieved. Electrochemistry using a suitable electrode as the electron source provides a promising method for delivering electrons to the P450 at a controlled electrode potential (ΔG for the electron-transfer reaction) [18], [19], [20], [21], in a manner that is species independent. In addition, this system can improve the rate-limiting step of the P450 reaction and is generally applicable to various kinds of P450. Although direct electron transfer from the electrode to P450 is desirable, it generally requires immobilization of the enzyme on the electrode surface [22], [23], [24], [25], which can cause the conformation of the P450 to change, thereby altering its properties/activity [26], [27], [28], [29]. In addition, the dioxygen molecule, one of the substrates of P450, can be reduced at the electrode surface to become reactive oxygen species (ROS) that deactivate the P450 on the electrode surface [30], [31]. Therefore, a mediated electron transfer (MET) system that uses mediator molecule(s) without P450 immobilization may be more favorable. Furthermore, the MET system can be used with cell-culture extracts that express P450, which avoids the laborious and time-consuming purification step in the enzyme-preparation process. MET electrochemistry for some P450 species has been reported [31], [32], [33], [34], [35]; however, it is affected by another oxygen-related problem, namely electron loss from the reduced mediator to the dissolved dioxygen, which decreases conversion efficiency [36]. Although some approaches toward solving this issue have been reported/suggested [37], [38], [39], it remains a general problem that, together with the ROS issue, hinders the development of efficient P450 electrocatalysis.

In the present study, we used Vdh as a P450 model and developed a MET system that solves the aforementioned oxygen-related problems. Mediators, additives, electrode surface modifications, electrode materials, and P450 sample conditions were examined. 25-Hydroxy VD3 was produced more efficiently under optimum conditions when compared to that formed by a conventional in vitro P450 conversion system that uses NADH as the electron source. Furthermore, we found that the present system works for E. coli cell extracts that express Vdh.

Section snippets

Expression and purification of recombinant Vdh

Constructions of expression plasmid for wild-type Vdh and two mutants, T107A and T70R/V156L/E216M/E384R (designated as K1), and overexpression of the Vdh was performed according to the previously described procedures [16], [40] with slight modification. Briefly, Escherichia coli BL21 CodonPlus (DE3)-RIL cells were pre-cultured in 100 mL Luria-Bertani (LB) medium at 37 °C overnight, and then inoculated to 900 mL LB medium containing 100 μM FeSO4 and 80 μg/mL 5-aminolevulinic acid. The

Effective mediators with less electron loss

We first tested phenosafranine (PSF) and safranine (SAF) as molecules that mediate electron transfer from the electrode to Vdh using gold and ITO electrodes with various surface modifications. Both PSF and SAF are useful mediators that supply electrons to some P450 species [32], [43]. However, no product was detected in the case of Vdh, which indicates that insufficient electrons were transferred to Vdh. Therefore, we used spinach ferredoxin (Fd) as an additional mediator to facilitate the

Conclusions

In summary, we explored an electrochemical electron supply system for P450 vitamin D3 hydroxylase, which efficiently produces 25-hydroxy-vitamin D3. Neutral red was found to be a suitable mediator that enables electrons to pass to Fd but with few passing to the oxygen, which decreases electron loss through side reactions. Modification of the electrode surface was also found to be crucial; the modified electrode surface prevents the deactivation of P450 by ROS. Under the optimum conditions in

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was partly supported by the JSPS KAKENHI Grant Numbers JP16K08218 and JP19K07024.

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