English

• Unspecific peroxygenases (UPOs, EC 1.11.2.1) is a kind of highly glycosylated thioheme enzyme, which belongs to the heme sulfate peroxygenase (HTP) superfamily [1]. At present, it is documented that UPOs mainly exist in Dikarya, Ascomycota, and Basidiomycota. Under mild conditions, UPOs are capable of catalyzing a variety of oxidations of inert C—H bonds with hydrogen peroxide (H₂O₂) as the only oxygen donor [2], such as hydroxylation [3], epoxidation of compounds containing C=C bonds [4], and sulfide sulfonation [5]. Therefore, UPOs are well-recognized as a potential enzyme for the oxidative functionalization of a wide range of substrates [6] in the fields of food processing [7], pharmaceutical production [8], and environmental protection [9].

  Among the types of reactions that UPOs can catalyze, the reaction that catalyzes the hydroxylation of C—H bonds of fatty acids to generate hydroxyl fatty acids is of great significance. Hydroxy fatty acids are saturated or unsaturated fatty acids containing single or multiple hydroxyl groups [10,11]. They have shown great potential in the synthesis of precursors of polyester fiber and polyamides [12,13], lactone and other flavor compounds [14], drug intermediates [15,16], surfactants, and emulsifiers [17]. Due to the limitations and drawbacks of traditional chemical synthesis, the synthesis of hydroxyl fatty acids via an enzymatic way has become a research hotspot in recent years [18]. And some UPOs have been successfully used in the synthesis of hydroxyl fatty acids [19–22].

  It has been sufficiently demonstrated that UPOs are cofactor-independent enzymes catalyzing oxidative reactions with H₂O₂ as an oxygen donor. However, unfortunately, the formation of iron peroxide in high concentrations of H₂O₂ usually leads to collapsed active heme centers of UPOs due to their strong affinity for H₂O₂ [23]. The poor H₂O₂ tolerance of UPOs has become a bottleneck for their large-scale industrious applications. The combination of a sensitive H₂O₂ sensor and an automatical H₂O₂ feeding system has ever been proposed to solve this issue [24], but the development of this equipment requires unacceptable high costs. To maintain the stability and catalytic activity of UPOs, some in situ H₂O₂ generating methods have been established via photochemistry [25–28], electrochemistry [29–33], and multi-enzyme cascade [34–37]. High costs and complicated operations are also involved in these approaches, thereby hindering the further applications of UPOs. Despite the impressive advances in the H₂O₂ tolerance research of UPOs in the last department, it scenes that we have merely scratched the surface that far, instead of solving the problem from the protein structure level. Therefore, clarification of the relationship between structure and H₂O₂ tolerance of UPOs appears to be a potential way for the development of H₂O₂-resistant UPOs based on the molecular modification.

  The key to exploring the amino acid sites that affect H₂O₂ tolerance in UPO structure is to obtain protein crystals. Therefore, achieving heterologous expression of UPO and obtaining high-quality enzymes is a prerequisite for protein crystal preparation and analysis. But the heterologous expression of UPOs still faces challenges, since the discovery of the first UPO (AaeUPO) in 2004, only about 20 kinds of UPOs have been identified and achieved heterologous expression [38], and just a few protein crystal structures of UPOs have been resolved, including AaeUPO (pdb_2YOR) (UPO from Agrocybe aegerita) [39], the PaDa-Ⅰ mutant of AaeUPO (pdb_5OXU) [40], MroUPO (pdb_5FUK) (UPO from Marasmius rotula), HspUPO (pdb_7O1R) (UPO from Hypoxylon sp. EC38) [41] and rCviUPO (pdb_7ZCL) (UPO from Collariella virescens) [42]. Nevertheless, the relationship between structure and H₂O₂ tolerance of UPOs has been hardly elucidated due to the lack of crystal structure of more UPO proteins. Therefore, the rule-of-thumb for the H₂O₂ tolerance-oriented molecular modification of UPOs is still lacking.

  To (partially) address this gap, we chose the UPO from Daldinia caldariorum (rDcaUPO) for expression and further investigate the relationship between protein structure and H₂O₂ tolerance in the present study. rDcaUPO is the second active recombinant UPOs heterologously expressed in E. coli and has great potential in catalyzing the hydroxylation reaction of fatty acids [43]. The molecular modification of rDcaUPO is feasible due to its remarkably higher production level than MroUPO using the same expression system [44]. In this study, we heterologously expressed the rDcaUPO in the E. coli prokaryotic system. We analyzed the amino acid composition near the active center of rDcaUPO and identified three crucial sites that might affect its H₂O₂ tolerance. Nine mutants were designed to elucidate the relationship between these sites and H₂O₂ tolerance. Gratifyingly, we show that the mutant rDcaUPO-A161C, exhibited significantly improved H₂O₂ tolerance and higher catalytic efficiency for the enzymatic production of hydroxyl fatty acids compared with the wild type. The mechanism for H₂O₂-tolerance improvement of rDcaUPO-A161C was preliminarily analyzed based on its crystal structure. This study would provide an insight into the relationship between structure and H₂O₂ tolerance of UPOs.

  Compared with the "short" UPOs with reported structures, the protein sequence similarity between rDcaUPO and HspUPO is as high as 67% (Fig. 1 and Table S1 in Supporting information). The phylogenetic analysis of rDcaUPO showed that rDcaUPO was clustered together with HspUPO in the phylogenetic tree, suggesting that rDcaUPO might have the same functional properties (Fig. S1 in Supporting information). This observation also provided a theoretical basis for the subsequent template selection for rDcaUPO homologous modeling.

  Figure 1

  

  Figure 1.  Structure-based amino acid sequence alignment of the mature rDcaUPO with the template HspUPO. Residues in the conserved PCP and EHD motif of the UPOs family were green frames.

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  The rDcaUPO wild type and its mutants were expressed in E. coli BL21 (DE3). The crude enzyme solution was eluted by a Ni affinity chromatography column and the purity of desalted enzyme solution met the requirements of subsequent experiments (Fig. S2 in Supporting information). The biochemical properties of rDcaUPO were determined by the NBD method (Table S2 and Fig. S3 in Supporting information). The optimum temperature of rDcaUPO was 30 ℃ and the optimum pH was 6, suggesting the stability in a neutral and partial acid environment. Metal ions Fe³⁺ and Fe²⁺ promoted the enzyme activity of rDcaUPO, and rDcaUPO was not sensitive to most organic solvents and metal ions.

  Usually, each UPO-catalyzed reaction requires two H₂O₂ molecules. Karich et al. found that the decomposition process of the second H₂O₂ is similar to the peroxidase reaction of UPOs [45]. Cpd-Ⅰ extracts a hydrogen atom from the second H₂O₂ to form Cpd-Ⅱ. In heme-dependent catalase, distal histidine (His56) or asparagine (Asn129) stabilizes the H₂O₂ free radical (HOO^•) through hydrogen bonding. The excess H₂O₂ will lead to the formation of Cpd-Ⅲ (iron superoxide complex = [Fe^Ⅲ-O²⁻]). When Cpd-Ⅲ encounters extra H₂O₂, the formed hydroxyl radical (HO^•) will eventually induce the following formation of biliverdin via the "self-hydroxylation" of heme, thereby inactivating UPOs [45].

  Therefore, to protect the active "heme" center from excess H₂O₂ molecules and decelerate the heme "bleaching" by formed HO^•, some reductive amino acids were introduced near the catalytic center of the wild type rDcaUPO (Fig. 2A). Based on the redox properties of amino acids near the catalytic active center in the tertiary structure of rDcaUPO, the mutation targets were selected and replaced with some reduced amino acids (Fig. 2B). The rDcaUPO mutants include L59R, L59S, L59C, L86F, L86R, A161R, A161S, A161C, and A161K.

  Figure 2

  

  Figure 2.  Selection of amino acid key sites in the region near the active center (green) of rDcaUPO. (A) The overall structure of the rDcaUPO wild type. (B) Amino acid key sites of the active center of the rDcaUPO wild type.

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  To detect the effect of mutants on heme protection, firstly, the optimal H₂O₂ concentration of the rDcaUPO wild type and mutants were compared using NBD as substrate. As shown in Fig. 3A, the rDcaUPO wild type and mutants L59R, L86F, A161R, A161S, and A161C showed an optimum H₂O₂ concentration of 10 mmol/L, whereas the optimum H₂O₂ concentration of A161K decreased to 5 mmol/L. Therefore, mutations of these key amino acids changed the optimal H₂O₂ concentration of rDcaUPO.

  Figure 3

  

  Figure 3.  The optimal H₂O₂ concentrations and H₂O₂ stability of the rDcaUPO wild type and mutants. (A) Optimal H₂O₂ concentration. (B) The H₂O₂ stability under 10 mmol/L H₂O₂. (C) The H₂O₂ stability under 20 mmol/L H₂O₂. (D) The H₂O₂ stability under 50 mmol/L H₂O₂. (E) The H₂O₂ stability under 100 mmol/L H₂O₂. Values were means ± standard deviation from three independent experiments.

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  The H₂O₂ tolerance of the rDcaUPO wild type and mutants was further studied at different H₂O₂ concentrations. As shown in Figs. 3B–E and Table S3 in Supporting information, the H₂O₂ tolerance of these mutants changed compared with the wild type under different H₂O₂ concentrations (10, 20, 50, and 100 mmol/L). The residual enzyme activities of L59R, L86F, and A161K could not be detected after incubation with 10 mmol/L H₂O₂ for 4 h. The rDcaUPO mutants with mutations at A161 using amino acids of increased reducibility exhibited increased H₂O₂-tolerance. Compared with the wild type, the relative enzyme activity of A161K was 117%. Notably, the mutant rDcaUPO-A161C showed a higher H₂O₂ tolerance compared with other mutants. After incubation in 10 mmol/L H₂O₂, the half-life of enzyme activity was increased from 2.5 h to 12.5 h. After incubation in a high concentration of H₂O₂ (100 mmol/L), the half-life of enzyme activity was still more than 2 h, and the relative enzyme activity increased by 64%. The introduction of reductive amino acids near the active center of rDcaUPO contributed greatly to improved H₂O₂ tolerance.

  To further mechanistically explain the improved H₂O₂ tolerance of rDcaUPO-A161C, the crystal of rDcaUPO-A161C was prepared for structural analysis (Fig. S4 in Supporting information). The crystal structure of the ligand-free rDcaUPO-A161C was obtained at 1.47 Å resolution by the molecular replacement (MR) method. The data collection and refinement statistics are shown in Table S4 (Supporting information). The three-dimensional structure of rDcaUPO-A161C was uploaded to the PDB database (pdb_8IAG). The overall structure of rDcaUPO-A161C monomers (Fig. 4A) is compact and spherical. It consists of residues from Ala1 to Thr224, displaying clearly defined electron density together with one heme molecule (CPK sticks with Fe³⁺ as a red stick) and one Mg²⁺ ion (green sphere). The overall fold of rDcaUPO-A161C mutant contains a total of 10 α-helixes (cyan) and a series of ordered rings, the combination of which form a single asymmetric spherical structure. α-Helices include two short α4 and α9 helices (L77-S81 and Y90-L92) (Fig. 4A). Moreover, the two conserved domains near heme form an active site with the catalytic PCP regions (Pro16, Cys17, Pro18 residues) and EHD regions (Glu86, His87, Asp88 residues) (Fig. 4B). The heme co-factor is embedded within helices with the thiolate sulfur of Cys-17 coordinating with the heme iron. This pattern is attributed to the highly conserved "Peroxidase_2" domain (National Center for Biotechnology Information (NCBI) blastp) of chloroperoxidases [46,47]. Figs. 4C and D showed the surface pattern of the overall structure of rDcaUPO-A161C. The shape of the distal matrix binding bag is similar to the truncated cone. The entry channel directly contacts the Fe³⁺ ion above the distal heme.

  Figure 4

  

  Figure 4.  Structures of mutant rDcaUPO-A161C. (A) The overall structure of the mutant rDcaUPO-A161C. (B) Active sites of the mutant rDcaUPO-A161C. (C) Surface display of the mutant rDcaUPO-A161C. (D) Pocket of the mutant rDcaUPO-A161C.

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  The structural characterization showed that rDcaUPO-A161C has a similar three-dimensional structure to two "short" UPOs, i.e. HspUPO and rCviUPO (Fig. 5A), with α helix being the primary structure. The structural difference is that HspUPO contains two β-sheets (I143-D145 and T195-P197). Similarly, the α-helix includes two short α4 (P72-K74) and α6 (Y99-H102) of rCviUPO, which were not found in rDcaUPO-A161. The structural superposition showed that the active regions of PCP and EHD are highly conserved in the "short UPO family". In rDcaUPO-A161C, in addition to the conserved amino acids of the active site, the heme pocket includes residues arranged on the inside of the cavity, such as Leu86, Met19, Leu59, Cys161, and Glu158 (Fig. 5B). Mg²⁺ ion seem to be coordinated by His87 (backbone) carbonyl, two water molecules, carboxyl groups of Glu86, and a heme ring. In this structure, Mg²⁺ ion appear to anchor the heme cofactor in the central pocket of UPOs. In the same spatial position of HspUPO (Fig. 5C), the amino acid residues around the heme pocket are the same as those of rDcaUPO-A161C, which is consistent with the amino acid sequence similarity (67%) of these two proteins. In the heme pocket of rCviUPO, consistent with rDcaUPO-A161C, the heme iron is coordinated by the mercaptan side chain of Cys19 towards the lower side (Fig. 5D). The differences between the residues around the cofactor rDcaUPO (cyan) and rCviUPO (yellow) are that the equivalent positions of Leu86, Leu59, and Met19 in rDcaUPO-A161C become Phe88, Ile61, and Ala21 in rCviUPO, thus reducing the entrance to the channel.

  Figure 5

  

  Figure 5.  Stereo-view of the superimposition and the comparison of heme pocket residues (top) and access channels (bottom) between mutant rDcaUPO-A161C and other UPOs. (A) The overall structure comparison. (B) Active site key amino acids of mutant rDcaUPO-A161C. (C) The active site differences between mutant rDcaUPO-A161C (cyan) and HspUPO (gray). (D) The active site differences between mutant rDcaUPO-A161C (cyan) and rCviUPO (yellow). (E) Superposition of mutant rDcaUPO-A161C and wild type overall structure. (F) Major differences between mutant rDcaUPO-A161C and wild type (mutant in blue, wild type in pink).

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  The structure of rDcaUPO-A161C was regarded as a template to reconstruct the three-dimensional structure of the rDcaUPO wild type. To explore the improvement of H₂O₂ tolerance by rDcaUPO-A161C, the structure of rDcaUPO-A161C was compared with the rDcaUPO wild type (Fig. 5E). The conserved regions of PCP and EHD are located around an exposed heme in the catalytic center of the mutant (Fig. 5F). The results showed that the positions of heme in its active center and crucial amino acids including P16, C17, P18, and E87, H88, and D89 contributed to the unchanged catalytic oxidation function.

  The active site of UPOs is a conical closed channel. Cys acts as a heme ligand coordinated by the sulfhydryl side chain to stabilize the entire active site [26]. The Cys17 was used as the ligand for stabilizing the heme pocket of rDcaUPO-A161C in this study. The introduction of Cys near the upper pocket of the heme, to some extent, seemed to increase the stability of the heme pocket.

  Further structural analysis showed that the distance between the SH motif of the mutant Cys at position 161 and the C atom of the hydroxylation site on heme is 5.98 Å. In comparison, the distance of the C atom of the rDcaUPO wild type Ala is 6.97 Å (Fig. 5F). The -SH motif of the Cys of rDcaUPO-A161C is closer to the oxidation site above the heme than Ala of the wild type. It is speculated that the 161C position of rDcaUPO-A161C would be the initial reaction site when exposed to excessive hydroxyl radicals, thus delaying the bleaching process of heme. As a result, the inactivation process of rDcaUPO-A161C would be indirectly slowed down. This assumption is in agreement with the observation that rDcaUPO-A161C showed robust H₂O₂-tolerance.

  The catalytic oxidations of C8-C12 saturated straight-chain fatty acids by rDcaUPO and rDcaUPO-A161C were investigated. As shown in Table 1, rDcaUPO and rDcaUPO-A161C showed high hydroxylation reaction efficiency on the ω−1 position of C8-C12 fatty acids. Both rDcaUPO and rDcaUPO-A161C preferred to catalyze the hydroxylation of lauric acid. The concentration of ω−1 hydroxylated lauric acid produced by rDcaUPO-A161C was 0.38 mmol/L, which was higher than that of the wild-type.

  Table 1

  

  Table 1.  The catalytic reaction of rDcaUPO and rDcaUPO-A161C on fatty acid substrates with different chain length.

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  In conclusion, we reported a structure-guided enzyme engineering approach for improving the H₂O₂-tolerance of UPOs. Using this approach, we designed a mutant, i.e., rDcaUPO-A161C, which exhibited significantly enhanced H₂O₂-tolerance, enabling the hydroxylation of C8-C12 saturated straight-chain fatty acids. The structure-H₂O₂-tolerance relationship reported in this study would provide an insight into the design of function-targeted UPOs by structure-guided engineering.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationship that could have appeared to influence the work reported in this paper. Guangdong Youmei Institute of Intelligent Bio-manufacturing Co., Ltd. provides some experimental instruments for testing during the whole experiment process. The company will not have any conflict of interest.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (No. 32001633), the Key Program of Natural Science Foundation of China (No. 31930084), and Guangzhou Science and technology planning project (No. 202102020370).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108701.

  
  
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• 

  Figure 1  Structure-based amino acid sequence alignment of the mature rDcaUPO with the template HspUPO. Residues in the conserved PCP and EHD motif of the UPOs family were green frames.

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  Figure 2  Selection of amino acid key sites in the region near the active center (green) of rDcaUPO. (A) The overall structure of the rDcaUPO wild type. (B) Amino acid key sites of the active center of the rDcaUPO wild type.

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  Figure 3  The optimal H₂O₂ concentrations and H₂O₂ stability of the rDcaUPO wild type and mutants. (A) Optimal H₂O₂ concentration. (B) The H₂O₂ stability under 10 mmol/L H₂O₂. (C) The H₂O₂ stability under 20 mmol/L H₂O₂. (D) The H₂O₂ stability under 50 mmol/L H₂O₂. (E) The H₂O₂ stability under 100 mmol/L H₂O₂. Values were means ± standard deviation from three independent experiments.

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  Figure 4  Structures of mutant rDcaUPO-A161C. (A) The overall structure of the mutant rDcaUPO-A161C. (B) Active sites of the mutant rDcaUPO-A161C. (C) Surface display of the mutant rDcaUPO-A161C. (D) Pocket of the mutant rDcaUPO-A161C.

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  Figure 5  Stereo-view of the superimposition and the comparison of heme pocket residues (top) and access channels (bottom) between mutant rDcaUPO-A161C and other UPOs. (A) The overall structure comparison. (B) Active site key amino acids of mutant rDcaUPO-A161C. (C) The active site differences between mutant rDcaUPO-A161C (cyan) and HspUPO (gray). (D) The active site differences between mutant rDcaUPO-A161C (cyan) and rCviUPO (yellow). (E) Superposition of mutant rDcaUPO-A161C and wild type overall structure. (F) Major differences between mutant rDcaUPO-A161C and wild type (mutant in blue, wild type in pink).

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  Table 1.  The catalytic reaction of rDcaUPO and rDcaUPO-A161C on fatty acid substrates with different chain length.

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