Characterization of high-H2O2-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation

The crystal structure of CYP105D18 and its unique structural features for papaverine N-oxidation are presented.


Introduction
Cytochrome P450 (CYP) enzymes comprise a large superfamily of heme b-containing enzymes expressed in a variety of species. These enzymes catalyze diverse reactions, including hydroxylation, alcohol and carbonyl oxidation, epoxidation, demethylation, N-oxidation, ring cleavage, coupling, and formation of a vast number of endogenous and exogenous compounds, such as steroids, fatty acids, xenobiotics and other drugs (Isin & Guengerich, 2007). CYPs are involved in regioand stereo-specific reactions with substrate flexibility, making them suitable targets for biotechnological applications (Ciaramella et al., 2020). The complex mechanism of CYP reactions involves specific redox partners and costly cofactors, thereby limiting the broad utility of CYPs in nature. However, some CYP monooxygenases and peroxygenases support the shunt pathway using an oxygen surrogate system such as hydrogen peroxide (H 2 O 2 ) and organic surrogates such as cumene hydroperoxide or iodosylbenzene for efficient catalysis of different substrates (Wei et al., 2019;Munro et al., 2018). Implementation of this system may be economically viable, with some P450s evolving to use H 2 O 2, but is typically inefficient and may lead to heme oxidation and enzyme destabilization or inactivation (Ciaramella et al., 2020;Munro et al., 2018). In addition, the shape and size of the substratebinding pocket may hinder the formation of highly reactive compounds using the shunt pathway for efficient catalysis (Ciaramella et al., 2020;Wei et al., 2019).
The general ability of CYPs to utilize H 2 O 2 has been demonstrated for many human CYPs and bacterial CYPs. Human CYP1A2 supports H 2 O 2 shunting for efficient oxidation of aromatic amines (Anari et al., 1997), and CYP3A4 and CYP2D6 can convert pinacidil (a vasodilator drug) into its corresponding amine using H 2 O 2 (Zhang et al., 2002). The major human drug-metabolizing CYPs, i.e. CYP2C9, CYP2C19, CYP2D6 and CYP1A2, can also support different oxygen surrogate systems, and their catalysis function is based on the various substrates used (Strohmaier et al., 2020). Typically, members of the bacterial CYP152 family, including P450BS, P450SP and OleTJe, have been shown to utilize H 2 O 2 for efficient fatty acid hydroxylation (Munro et al., 2018).
Papaverine, an isoquinoline alkaloid present in opium, causes smooth muscle relaxation and has been used to treat vasospasm. Moreover, papaverine has been administered as an alternative to nonsteroidal anti-inflammatory drugs in some patients to treat renal colic pain, migraine headaches, schizophrenia, cancers and even viral infections (Shen et al., 2019;Aggarwal et al., 2020). Despite the medicinal importance of papaverine, this compound has many adverse effects. However, modified papaverine may have better efficacy and reduced cytotoxicity and can easily be metabolized by the body. Chemical modification of papaverine for C-H functionalization, N-oxidation (Egbewande et al., 2019) and Odemethylation has been reported (Brossi & Teitel, 1970). These classical approaches are time-consuming and costly and may suffer from poor regio-selectivity compared with enzymecatalyzed reactions. Shen et al. (2019) reported an efficient biocatalyst, CYP105D1, for the 6-O-demethylation of papaverine.
The CYP105 family is involved in biosynthetic pathways of secondary metabolites and biotransformation of xenobiotic compounds. For example, CYP105D7 from Streptomyces avermitilis catalyzes the hydration of 1-deoxypentalenic acid at the C1 position, isoflavone and daidzein at the 3 position and diclofenac at the C4 0 position (Takamatsu et al., 2011;Pandey et al., 2010;Xu et al., 2015). Additionally, CYP105A1 from Streptomyces griseous converts vitamin D 3 (VD 3 ) to 1,25dihydroxy vitamin D3, a hormonally active form, via two sequential hydroxylations (O'Keefe et al., 1988;Hayashi et al., 2008). CYP105P1 and CYP105D6 are also involved in the biosynthesis of filipin (Xu et al., 2010). Although the CYP105 family has diverse functions, all members share a common CYP structure within a relative mean squared deviation of 2 Å , indicating that further close structural investigation is required to determine the regio-and/or stereo-specific substrate recognition and chemistry (Lee et al., 2016).
In this study, we aimed to determine biochemical and structural information for proteins in the CYP105 family. We studied CYP, annotated as CYP105D18, from Streptomyces laurentii. We also determined the substrate-free and papaverine-complexed crystal structures of CYP105D18 at resolutions of 1.7 and 2.0 Å , respectively. To the best of our knowledge, this is the first report of an N-oxidation reaction using bacterial CYP.

Functional characterization of CYP105D18
Phylogenetic tree analysis of CYP105D18 revealed that it had the closest relationships with functionally characterized CYP105D7 (71.1%) and CYP105D1 (64.9%; Fig. S1 of the supporting information). CYP105D1 was well characterized for biotransformation of isoquinoline alkaloids (Shen et al., 2019). So, we speculated that this protein is involved in the biotransformation of different isoquinoline alkaloids (e.g. papaverine, THP, berberine and palmatine). CYP105D18 was cloned, overexpressed and purified. The theoretical molecular weight was 44.3 kDa, and a single band at $50 kDa (with a His-tag from pET28) was obtained by sodium dodecyl sulfatepolyacrylamide gel electrophoresis [ Fig. 1 (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purification of CYP105D18 [1, crude extract; 2, insoluble fraction; 3, marker (kDa); 4, 10 mM imidazole fraction; 5, 100 mM first fraction; 6, 100 mM second fraction and 7, 500 mM imidazole fraction]. The 100 mM imidazole second fraction (sample 6) was concentrated for in vitro reaction. The molecular weight of CYP105D18 is 44.3 kDa, and the protein was overexpressed using the pET28 vector in the C41 host. (b) Dithionite reduced the CO-bound form of CYP105D18.
CYP105D18 showed a Soret peak at 418 nm in an oxidized form. The carbon-monoxide-bound and dithionite-reduced form of CYP105D18 exhibited maximum absorption at a wavelength of 449 nm, one of the important spectral characteristics of CYP450 [ Fig. 1(b)]. The purity of a CYP can be evaluated from its R z value, calculated as the ratio of absorbance (A) at max of the Soret band to the A value at 280 nm. The purified enzyme had an R z value of 1.45, indicating high purity (Lee et al., 2016). The redox partner proteins Pdx and PdR were expressed and purified to single bands corresponding to 11.4 and 45.6 kDa, respectively (Fig. S2). The four isoquinoline alkaloids with different structures (listed in Fig.  S3) were screened for in vitro biotransformation by CYP105D18. There was no in vitro biotransformation by CYP105D18 for papaverine, palmatine, THP or berberine using the redox partners Pdx/PdR from the P450cam system and/or spinach Fdx/FdR (data not shown). CYP105D18 supported the N-oxidation of papaverine and hydroxylation of tetrahydropalmatine using the oxygen surrogate H 2 O 2 . Highperformance liquid chromatography (HPLC) analysis of papaverine revealed product formation at a retention time of 18.2 min, equivalent to hydroxylation/N-oxidation in liquid chromatography-mass spectrometry (LC-MS) analysis [ Fig.  2(a)]. The HPLC results showed an additional peak at a retention time of 22.2 min, which could not be identified because of the poor degree of ionization by LC-electrospray ionization-MS. The major product was purified and characterized by nuclear magnetic resonance analysis as papaverine N-oxide (Fig. S4). Similarly, the tetrahydropalmatine product catalyzed by CYP105D18 in the presence of H 2 O 2 showed a single peak at a retention time of 13.5 min in the HPLC analysis [ Fig. 2(b)]. The LC-MS data revealed the mono-hydroxylation (mass, 372.179 g mol À1 ) of tetrahydropalmatine. The maximum product conversion for tetrahydropalmatine under optimum conditions was less than 12%; therefore, we did not purify the product for structural analysis. The results showed no support from H 2 O 2 as an oxygen surrogate partner for biotransformation of berberine and palmatine by CYP105D18.
To support the in vitro biotransformation of papaverine by CYP105D18, we performed the substrate-binding experiment. Among the four substrates, only papaverine induced the spectral shift, which was at 422 nm ( Fig. S5). Even though THP was catalyzed by CYP105D18 using H 2 O 2 as cosubstrate, there was no spin-shift observed. The binding of 4-(pyridine-3-yl) benzoic acid and 4-(pyridine-2-yl) benzoic acid to CYP199A4 exhibits type II UV-vis spectral changes showing a greater shift in the Soret wavelength (422 versus 424 nm) (Podgorski et al., 2020). Papaverine was also modified in the pyridine ring (N-oxidation) by CYP105D18 and exhibits a similar pattern of spectral shift at 422 nm. In addition, there was a spectral difference in the band between substratebound and unbound forms. Papaverine-bound spectra showed a Soret band at 322 nm, which was not observed in the binding of 4-(pyridine-3-yl) benzoic acid and 4-(pyridine-2-yl) benzoic acid to CYP199A4 (Podgorski et al., 2020). This is the first report of N-oxidation of a compound by bacterial CYP. However, human CYP has been reported to catalyze the N-oxidation of drugs during drug modification by liver microsomes. CYP2E1 is the main microsomal enzyme involved in the N-oxidation of nicotinamide (Real et al., 2013), and CYP2C9, CYP2C19 and CYP3A4 exhibit different affinities for N-oxidation of the antifungal drug voriconazole during its metabolism in liver microsomes (Hyland et al., 2003). In addition, the fungal aromatic peroxygenase (AaP) was reported to catalyze the N-oxygenation of pyridine and related compounds (Ullrich et al., 2008). Nitrogen-rich heterocyclic compounds are used to treat various human diseases, and these motifs are found in many isoquinoline alkaloid drugs (Egbewande et al., 2019). The N-oxide deriva- HPLC chromatogram and LC-MS spectra (inside the box) for (a) papaverine and (b) tetrahydropalmatine. The reaction (above) was performed with 400 mM substrate and 3 mM CYP105D18 in the presence of 40 mM H 2 O 2 for 1 h. A similar reaction condition was used for the control (below), with the use of 3 mM heat-deactivated CYP105D18 (positive control) and without the CYP105D18 (negative control). The positive and negative controls showed a similar HPLC chromatogram without any product formation. tives of isoquinoline alkaloids are structurally and functionally related to the original compound and are reported to have good pharmacological properties (Dembitsky et al., 2015). Bremner & Wiriyachitra (1973) reported the chemical synthesis of papaverine N-oxide from papaverine using chloroform and m-chloroperbenzoic acid. The photochemical synthesis of papaverine N-oxide was reported by Girreser et al. (2003). However, chemical and photochemical methods are timeconsuming and exhibit poor yields, as reported previously. For example, papaverine was treated with aqueous H 2 O 2 at 70 C for up to 10 h to produce papaverine N-oxide (Girreser et al., 2003). In contrast, we showed that CYP105D18, in the presence of a stoichiometric amount of H 2 O 2 , efficiently transformed papaverine to papaverine N-oxide, providing important insight into chemical methods.  functioned in the presence of a high concentration of H 2 O 2 , as reported in our previous work (Dangi et al., 2018). The stability of heme also plays a crucial role in the efficient catalysis of the CYP. For example, P450116B5hd showed higher tolerance than P450BMP (Ciaramella et al., 2020 Fig. 3(h). A product conversion rate of 70% was observed with 5 mM H 2 O 2 . The product conversion was more than 95% between 40 and 200 mM H 2 O 2 . The heme dissociation constant was less than 0.014 min À1 , and product conversion was greater than 97% using 40 mM H 2 O 2 within 30 min. Therefore, 40 mM H 2 O 2 was used as the optimum concentration to evaluate the enzyme kinetics. The product conversion decreased sharply when the the H 2 O 2 concentration was greater than 200 mM, and more than 10% conversion was observed in the presence of 800 mM H 2 O 2 .
Time-dependent in vitro conversion of papaverine N-oxide by CYP105D18 is shown in Fig. 4(a). The K m and k cat values for papaverine were estimated to be 213 AE 26 mM and 5 AE 0.3 min À1 , respectively [ Fig. 4(b)]. Similarly, K m and k cat values for tetrahydropalmatine were 75 AE 15 mM and 0.13 AE 0.01 min À1 , respectively [ Fig. 4(c)]. The catalytic efficiency (k cat /K m ) for papaverine was much higher (1.43 s À1 mM À1 ) in comparison with tetrahydropalmatine (0.11 s À1 mM À1 ).

Overall substrate-free structure of CYP105D18
Structural investigation of CYP105D18 was conducted to obtain detailed structural information related to the active site. Crystallization experiments for full-length CYP105D18 yielded crystals in the space group C2 diffracting to 1.7 Å . A final model was obtained with R factor and R free values of 0.16 and 0.21, respectively, and a Molprobity score of 1.48 (Williams et al., 2018). A Ramachandran plot analysis of the refined model showed that 96.55 and 0% of non-glycine residues were in favored regions and outliers, respectively. The final model of CYP105D18 exhibited electron density for most of the molecule, except for the N-terminus (amino acids 1-3) and a loop containing residues 73-85, which connected B and C [dotted line in Fig. 5(a)]. The asymmetric unit had one molecule of CYP105D18. Consistent with this observation, size exclusion chromatography analysis of CYP105D18 revealed that the protein existed as a monomer in solution (Fig. S6). The coordinates were deposited in the Protein Data Bank under entry 7di3 (substrate-free structure). Data collection and refinement statistics are summarized in Table  S4 of the supporting information.
As observed in the structures of the CYP105 family, CYP105D18 can be divided into two units: -sheet-containing units and helix-rich units composed of helices C-J [ Fig.  5(a)]. A long I was located in the middle between the two units and connected two units. CYP105D18 contained a heme surrounded by loops from the -sheet-containing unit and C, I and L from the helix-rich unit. The last C-terminus long loop starting from Lue366 covered the concave active site. As shown in the phylogenetic tree, CYP105D18 had the highest homology with the CYP105D subfamily, including CYP105D4, CYP105D7, CYP105D8 and CYP105D1. Consistent with this, structural similarities generated from the DALI server (http:// ekhidna2.biocenter.helsinki.fi/dali/) showed high similarity with structures of CYP105D7 and CYP105D6 (PetD) from S. avermitilis (Z-scores: 53.7 and 53.0, respectively; Table 2) (Xu et al., 2015(Xu et al., , 2010. CYP105D18 forms a deep, concave and funnel-like active site located in the center between the -sheet and helix-rich units; at the bottom of the active site, the heme plane is located vertically with respect to the active site funnel (Fig. 5). The active site funnel shape is composed of a B/C loop (residues 173-187), turn region between F and G, and C-terminal loop (residues 336-396). Although the CYP105 family shares high structural homology but has diverse substrates, residues      Multiple sequence alignment with other homologous CYPs. CYP105D18 from S. laurentii, CYP105D7 and CYP105D6/PteD from Streptomyces sp., CYP105D6 from S. avermitilis, CYP105N1 from S. coelicolor, CYP105A1 from S. griseolus, CYP105AB3 from Nonomuraea recticatena, GfsF from S. graminofaciens, CYP105AS1 from Amycolatopsis orientalis, and CYP105P1 from S. avermitilis. Secondary structural elements are shown above the aligned sequences and colored according to the scheme in Fig. 5(a). Regions for the active site are marked with orange sticks, and conserved residues at the B/C loop and turn of F-G are marked by red circles.
from these three regions are barely conserved [ Fig. 5(b)], indicating that these regions are essential for substrate selectivity for the CYP family. A previous study of CYP105D7 indicated that the arginine residues (Arg70, Arg88 and Arg190) in the B/C loop and G of the substrate-binding pocket are conserved in the CYP105 family and may be important residues for substrate recognition (Xu et al., 2015). Unlike CYP105D7, CYP105D18 contains Ser63 and Phe184, corresponding to Arg70 and Arg190 of CYP105D7. This suggests that the residual recognition of the substrate at the active site of CYPs is more complicated and varied.

Structural feature for the H 2 O 2 stability of CYP105D18
Previous studies have reported that human P450 1A2 is thiol-insensitive to H 2 O 2 . The molecular dynamics simulation using rabbit P450 4B1 as a model suggested that the Gln451 of P450 4B1 is an essential residue for the thiol sensitivity of P450 (Albertolle et al., 2019). Gln451 in P450 4B1 showed heme thiolate and sulfenic acid-dependent open and closed conformations, respectively. These conformational changes alter the solvent accessibility of heme-thiolate Cys488. Once the Cys488 was oxidized to sulfenic acid, the Gln451 forms an NH-bond with Phe441 for closed conformation, limiting the accessibility of oxidizing agents to the protein (Albertolle et al., 2019). Consistent with this, H 2 O 2 -stable CYP105D18 shows high sequential and structural similarity with P450 4B1. Across from the heme molecule are Phe338 and Gln348, which correspond to Phe441 and Gln451 of P450 4B1 and show a closed conformation. Notably, the substrate-free CYP105D18 shows a small blob on the heme molecules, as depicted in Fig.  6, indicating that the heme of CYP105D18 is oxidized. A similar mechanism with the P450 4B1 access region to the heme region of CYP105D18 may involve conformational changes upon oxidation, and this closed conformation probably results in the H 2 O 2 resistance of CYP105D18 (Fig. 6).

Comparison of substrate-binding pockets between substrate-free and papaverine complexes with CYP105D18
In addition to the substrate-free structure of CYP105D18, we determined the papaverine complex structure (PDB entry 7dls) to support papaverine N-oxidation and understand the substrate-binding mode and structural changes that occur upon substrate binding. The papaverine complex structure was superimposed with the substrate-free structure and showed an overall C relative mean squared deviation of 0.196 Å . Structural comparisons between the substrate-free and complex forms of CYP105D18s revealed that conformation changes occurred mainly in the B/C loop region. A flexible, less structured loop was observed in the B/C loop, and electron density for residues 73-84 was absent from the substrate-free structure, indicating that this region had a high degree of flexibility. However, the papaverine complex structure had a well defined electron density for the B/C loop and formed a short helix named B 0 , which has been previously observed in the diclofenac complex CYP105D7 structure (Xu et al., 2015). Although the B/C loop is a less conserved region, Phe68 and Pro69 are conserved within the CYP105 family. Phe68 is located at the starting point of the B/C loop and is deeply embedded by interactions with Val286 and Val288 from 4, which may act as a hinge with Pro69 for B/C loop movement. Consistent with this observation, the electron density after Pro69 was blurry with a high B-factor ($50 Å 2 ), whereas the electron density for Phe68 and Pro69 showed a sharp and a good fit with a low B-factor [$30 Å 2 ; Fig. 7(a)]. Taken together, these findings show that the B/C loop in the active site of the CYP105D18 structure was flexible and remained in an Superposition of CYP105D18 with H 2 O 2 -insensitive rabbit P450 4B1. Residues corresponding to Cys448, Phe441 and Gln451 of P450 4B1 are shown using sticks. The 2F o À F c electron density map for the heme and water molecule for CYP105D18 is shown as a gray mesh (contoured at the 2.0 level). The octane molecule from P450 4B1 is presented as a purple stick. open confirmation without substrate. Once the substrate bound to CYP105D18, the B/C loop formed a helix (B 0 ) with a closed conformation. This region is thought to be responsible for substrate binding and specificity (Lee et al., 2016;Xu et al., 2009).
Papaverine was buried within the internal hydrophobic cavity of CYP105D18 and was surrounded by residues Ala85, Leu87, Val231, Ala282, Val234 and Ile386, with no hydrophilic residues [ Fig. 7(b)]. The dimethodyisoquinoline group of papaverine was closely situated in the center of the heme, and dimethodyisoquinoline nitrogen was located toward the iron of the heme group. An electron-density blob corresponding to a water molecule between the isoquinoline nitrogen and heme iron was found (Fig. S7). The distance between the water and nitrogen was 3.2 Å , and that between the water and iron was 2.1 Å , indicating that the binding mode represented the true mode for N-oxidation (Fig. 8). The dimethodyisoquinoline and dimethoxy-phenyl groups were bent by approximately 90 , with a single carbon bond. This finding strongly supports our biochemical data demonstrating that CYP105D18 selectively transformed papaverine, although the overall biochemical characteristics of the substrates tested were similar because other isoquinoline alkaloids are linked using two single carbon bonds that cannot be bent.  To further support this observation, a molecular docking simulation was conducted with bendable substrates and nonbendable substrates. First, we used the papaverine model to check the reliability of the docking simulation. The simulated papaverine binds to the substrate-binding pocket of CYP105D18 with a similar binding mode to that of the co-crystallized structure (Fig. 9). The simulation results show that nitrogen of bendable chemicals has a closer distance (2.5-2.7 Å ) with water molecules interacting with iron of the heme molecule in CYP105D18 compared with those of non-bendable chemical compounds (4.4-8.6 Å ). Taken together, we suggest that the bending motion of the ligand may be the  Bending motion of papaverine in the substrate-binding site of CYP105D18. (a) Detailed binding mode of papaverine with CYP105D18. Papaverine is at the bottom of the active site. The sphere of water molecules (green) is between the dimethodyisoquinoline nitrogen and iron of the heme group. Papaverine and heme are colored yellow and magenta, respectively. (b) Close-up view of the binding pocket in (a). Cross-sectional images of CYP105D18 were generated using PyMol (Schrö dinger).

Figure 9
Comparison of bendable and non-bendable substrates. Co-crystallized papaverine and heme molecules with CYP105D18 are shown as yellow and orange sticks. Each potential substrate after the docking calculation was represented with sticks with different color codes. The distances between the water molecules interacting with heme in CYP105D18 and nitrogen are indicated in each figure. Docking experiments were performed using the Lamarckian genetic algorithm (LGA) method in Autodock Vina (Trott & Olson, 2010). critical feature in establishing the distance and coordinates during hydroxyl group transfer. Based on this analysis, we listed potential substrates for CYP105D18 in Fig. S8. Overall, this structural analysis supported the unique features of CYP105D18, including high flexibility at the active site entrance and substrate selectivity.

Conclusions
In this study, we reported a novel cytochrome P450 enzyme annotated as CYP105D18. We observed H 2 O 2 -mediated Noxidation of papaverine by this CYP and its high tolerance to H 2 O 2 for papaverine biotransformation. In addition, we investigated the crystal structure of CYP105D18, which suggested that high structural similarity is shared by the CYP105 family. Structural comparison of substrate-free CYP105D18 with a substrate-bound structure revealed high flexibility at the active site entrance comprising the B/C loop. In a papaverine-bound structure, papaverine adopted a bent structure that enabled correct alignment with the heme molecule for the N-oxidation reaction of papaverine. This finding explains why CYP105D18 had strong papaverine activity but weak or no activity for other isoquinoline alkaloids with non-bendable structures in our data. Taken together, our findings showed that this novel CYP exhibited high tolerance for H 2 O 2 and unique substrate specificity, warranting studies of substantial drug modifications for biotechnological applications.

Related literature
The following references are cited in the supporting information: Bhattarai et al. (2013); DeLano (2002)