Role of AcCO in the transformation from CHDM to CHDA
The synthesis of CHDA from its structural analog CHDM through a primary alcohol oxidation reaction can be catalyzed by AOX, ADH, and laccases. However, the regioselectivity of CHDM poses a major challenge for executing this reaction, as these enzymes could regioselectively oxidize one hydroxyl group of CHDM to produce the intermediate (HMCA), thereby changing the chemical properties and hindering further oxidation of another hydroxyl group (Additional file 1: Fig. S1). To address this challenge, we selected three different strains for each enzyme to evaluate the specific activity of those enzymes toward CHDA. Among them, the FAD-dependent AOX from A. cholorphenolicus (AcCO) and ADH from Sulfolobus solfataricus (SsADH) displayed the highest ability to produce CHDA (specific activity of 0.11 ± 0.026 and 0.094 ± 0.015 U·g−1, respectively) (Additional file 1: Tables S4–S6). However, SsADH requires an NAD+/cofactor-recycling system, and the equilibrium is unfavorable for the preparation of the aldehyde; therefore, AcCO was selected for further study, given that it uses oxygen (air) for cofactor regeneration and the reaction is irreversible (Scheme 1). Furthermore, AcCO can be heterologously expressed in E. coli [many other AOXs (Cleveland et al. 2021; Mathieu et al. 2020) and ADHs (Zhao et al. 2009) require eukaryotic expression systems] and the recombinant enzyme exhibited higher yield for the production of CHDA, with a ratio of CHDA to HMCA (RCHDA/HMCA) of 0.25 (Additional file 1: Figs. S2 and S3).
To increase the primary alcohol oxidation activity of AcCO, the expression conditions, including the induction temperature, inducer concentration, and induction time, were optimized. As shown in Figs. 1A–C, the specific activity of AcCO toward CHDA increased from 0.11 ± 0.026 U·g−1 to 0.29 ± 0.097 U·g−1 under the optimum conditions (16 °C for 2.5 h, 0.4 mM IPTG, and induction time of 16 h). Furthermore, the effect of organic solvents, buffer pH (6.5–8.5), temperature (20–35 °C), and organic solvent concentration [0–10% (v/v)] on CHDA production were investigated, and the results are shown in Figs. 1D–F and Additional file 1: Fig. S4. The yield of CHDA increased to 13.5% under the optimum conditions [30 g L−1 wet E. coli, pH 8.0, 100 mM air-saturated potassium phosphate buffer, 5% (v/v) DMSO, 0.1 g L−1 catalase, 30 °C for 12 h]. The yield of CHDA from CHDM slightly increased from 5.0 to 13.5%, although the titer and yield of HMCA from CHDM continued to increase from 1.0 mM to 1.4 mM and from 20.4 to 28.1%, respectively, resulting in an increase of the RCHDA/HMCA to 0.48. Therefore, we determined that further engineering of AcCO was required to increase the selectivity of CHDA and reduce the accumulation of HMCA. Toward this end, we explored the structure of AcCO and the catalytic mechanism of primary alcohol oxidation reaction.
The catalytic mechanism of AcCO
A homology model of AcCO was reconstructed using SWISS–MODEL (https://swissmodel.expasy.org/) based on the crystal structure of choline oxidase from A. globiformis (91% sequence identity) (Fig. 2A and Additional file 1: Fig. S5). According to the previous reports on AgCO, H466 plays an important role in the reaction as a catalytic base and FAD is involved in hydride (H−) transfer (Smitherman et al. 2015). Docking analysis using the AcCO structural model and CHDM (Fig. 2B) revealed that the contact between CHDM and FAD occurs through an O atom of the 1-Cα-OH, which is at a distance of 3.0 Å from the N(5) atom of FAD. Moreover, in the active center of AcCO, two hydrogen-bond interactions were identified between 1-Cα-OH of CHDM with the side-chain amide of N510 (2.5 Å) and the Nε2 atom of H466 (3.0 Å), as well as steric interactions between the C atom of CHDM with the side chains of V464 (3.6 Å) and H351 (3.7 Å), anchoring CHDM in the binding pocket. In this conformation, the cyclopentane portion of CHDM was almost parallel to the FAD isoalloxazine ring, which results in 1-Cα-OH of CHDM being retained on the inside of the binding pocket and close to the active center and 4-Cα-OH of CHDM extending toward to the outside of the binding pocket stay away from the catalytic center; the distance between the 4-Cα-OH with the N(5) atom of FAD and the Nε2 atom of H466 is 7.9 Å and 9.8 Å, respectively (Additional file 1: Fig. S6A). These findings indicated that the two Cα-OH groups of CHDM incompletely fit in the binding pocket, and the conformation of CHDM is not beneficial to produce HMCA. Given the accumulation of HMCA, we next analyzed the AcCO and HMCA docking model (Fig. 2C). Four hydrogen-bond interactions were identified between the 1-CHO with the N(5) atom of FAD (2.4 Å), the side-chain amide of N510 (3.3 Å), the Nε2 atom of H466 (3.3 Å), and the side-chain hydroxy of S101 (3.2 Å), as well as steric interactions between HMCA with the side chains of V464 (3.9 Å) and H351 (3.5 Å). In this conformation, although the cyclopentane plane of the HMCA generated an angle deflection [the angle (θ) between the 1-CHO of HMCA, the N(5) atom, and the N(10) atom of FAD (θN(10)-N(5)-(1-CHO)) shifted to 95° (the θN(10)-N(5)-(1-Cα-OH) is 124.1° in the WT-CHDM complex)], the 4-Cα-OH of HMCA was maintained toward the outside of the binding pocket (the distance between 4-Cα-OH of HMCA and the Nε2 atom of H466 was 10.0 Å) and the 1-CHO of HMCA was stably buried in the active center (Additional file 1: Fig. S6A, B). We deduced that it is difficult to further oxidize the 4-Cα-OH of HMCA due to the long catalytic distance from the active center, which causes the accumulation of HMCA. To verify this conjecture, MD simulation and QM/MM calculations were performed, the representative snapshot and the change of energy barrier were used to understand the molecular basis of the primary alcohol oxidation reaction.
As shown in Fig. 3A, based on the catalytic mechanism proposed by Gadda’s group (Smitherman et al. 2015; Gadda 2020), His466 should be deprotonated under basic conditions. The QM/MM optimized reactant complex shows that the deprotonated His466 forms a stable hydrogen bond with the Cα-OH of CHDM or HMCA. This result suggested that deprotonated His466 can serve as a basic catalyst during the transfer of the hydride (H−) from CHDM or HMCA to FAD. To verify the function of this residue, we mutated H466 to alanine, finding that H466A completely abolished the primary alcohol oxidation activity (Fig. 4). Thus, we inferred that AcCO catalyzed CHDM to CHDA which could be divided into two reaction steps: (I) reductive half-reaction, and (II) oxidative half-reaction. In the reductive half-reaction, the deprotonated histidine (H466) side chain attacks the 1-Cα-OH of CHDM and a proton from 1-Cα-OH of CHDM is transferred to the deprotonated histidine side chain (H466). Subsequently, the hydride (H−) in 1-Cα-H of CHDM is transferred to the N(5) of FAD isoalloxazine ring to form FADH−, this step involves a 13.5 kcal·mol−1 energy barrier (Fig. 3B). Finally, 1-Cα-OH of CHDM is oxidized to aldehyde, accompanied by HMCA synthesis. In the oxidative half-reaction, the hydride (H−) of C4α-peroxy in FADH− and a proton in H466 are transferred to the O-atom of oxygen, to regenerate FAD and release H2O2. Subsequently, 4-Cα-OH of HMCA is again attacked by H466, and the hydride (H−) is transferred from 4-Cα-H of HMCA to the N(5) of FAD isoalloxazine ring; this step involves a 20.2 kcal·mol−1 energy barrier and is accompanied by the synthesis of CHDA (Fig. 3B). In this catalytic mechanism, the second but most important step is the transfer of hydride (H−) from the CHDM and HMCA to the N(5) of FAD isoalloxazine ring. During this step, 1-Cα-H of CHDM executes the hydride (H−) transfer and to form HMCA, and 1-Cα-H of HMCA again transfers the hydride (H−) to FAD isoalloxazine ring to ultimately synthesize the final product CHDA. Therefore, the energy barrier of the second step is directly related to product formation, which determines the efficiency of the primary alcohol oxidation reaction.
However, the QM/MM optimized reactant complex showed that the hydride (H−) transfer of CHDM and HMCA leads to an energy barrier of 13.5 kcal·mol−1 and 20.2 kcal·mol−1 in the second step, respectively. Meanwhile, the energy barrier of HMCA is much higher than that of CHDM in the hydride (H−) transfer process, which makes it more difficult to oxidize 4-Cα-OH of HMCA to form CHDA. Therefore, we speculated that the energy barrier of this step is the key factor to achieve the low catalytic efficiency of CHDM and the accumulation of HMCA; and how to decrease the energy barrier of hydride (H−) transfer is the main issue. According to the representative snapshot of the MD simulation, however, the CHDM and HMCA anchored in the binding pocket represent an unfavorable catalytically active conformation, which is similar to the docking model. In this conformation, the distance between 1-Cα-H of CHDM and the N(5) atom of FAD (D(1-Cα-H)-N(5)FAD = 4.3 Å) and the distance between 4-Cα-H of HMCA and the N(5) atom of FAD (D(4-Cα-H)-N(5)FAD = 9.6 Å) were not suitable for hydride (H−) transfer, which may have limited the transfer of hydride (H−) and formed a high energy barrier (Fig. 6B, D). This result also confirmed our previous conjecture. Therefore, changing the catalytically active conformation to decrease the distance of hydride (H−) transfer was considered to be a potentially effective strategy to reduce the energy barrier.
Directed evolution of AcCO
Analysis of the interaction of the WT-CHDM and WT-HMCA complex showed that the N(5) atom of FAD, the side-chain amide of N510, and the Nε2 atom of H466 have hydrogen-bond interactions with 1-Cα-OH of CHDM and 1-CHO of HMCA, along with an extra hydrogen-bond interaction between 1-CHO of HMCA and S101. The residues H351 and V464 are close to the cyclopentane plane of CHDM and HMCA, which may produce steric interactions with the C atom of CHDM and HMCA (Additional file 1: Fig. S7A, B). Thus, it was speculated that these interactions would firmly fix 1-Cα-OH of CHDM and 1-CHO of HMCA to the active center and produce the steric hindrance to hinder the adjustment of the catalytically active conformation. Therefore, six residues (S101, H351, Y465, N510, I103, and V464) in a radius of 5 Å around 1-Cα-OH of CHDM and 1-CHO of HMCA were selected (Additional file 1: Fig. S8A, B) for engineering by NNK site-saturation mutagenesis to change the interactions between the enzyme and CHDM or HMCA. Among them, the activity of two variants, S101A and H351V, increased by 3.3-fold and 4.3-fold to produce CHDA, respectively. Subsequently, variant W2 (S101A/H351V), representing the combination of these two single variants, was constructed, which showed the activity toward CHDA was 2.43 ± 0.13 U·g−1, that was 8.3-fold higher than that of WT. As a result, the yield of CHDA increased to 25.6%, and the yield and titer of HMCA increased to 63.8% and 3.19 mM, respectively, leading to an overall decrease of the RCHDA/HMCA to 0.41 and thus a large accumulation of HMCA (Fig. 4, Table 1). The preliminary docking model of the variant W2-HMCA, it demonstrated that the hydrogen-bond interaction between 1-CHO of HMCA and A101 was broken by mutating S101 to A101 (the distance between residue 101 and HMCA increased to 5.1 Å), and when the H351 was mutated to V351, the distance between the side chains of V351 and the C atom of CHDM and HMCA increased to 5.1 Å and 4.9 Å. This shift altered the D(1-Cα-H)-N(5)FAD of CHDM to 2.4 Å, while the distance of nucleophilic attack (D(1-Cα-OH)-Nε2H466) was reduced to 3.0 Å, and the D(4-Cα-OH)-Nε2H466 and D(4-Cα-H)-N(5)FAD of HMCA shifted to 9.0 Å and 7.8 Å, respectively (Additional file 1: Fig. S9). Therefore, we speculated that the conformation was slightly adjusted with the change of interactions, but 4-Cα-OH of HMCA was still not attached to the correct location, so that it was not well fixed to the inside of the pocket, consequently hindering the further oxidation of 4-Cα-OH.
To address this limitation, we considered that changing the interactions between HMCA and the enzyme in the active center may be an efficient strategy to adjust the catalytically active conformation and reduce the accumulation of HMCA. Therefore, the site-screening range was expanded to 8 Å of 4-Cα-OH of HMCA based on variant W2. This change was expected to alter the interactions between 4-Cα-OH of HMCA with the surrounding residues to adjust the catalytically active conformation and consequently reduce the D(4-Cα-H)-N(5)FAD of HMCA. Toward this end, nine residues (W331, T463, N462, W61, F357, N378, Q329, V355, and M359) next to 4-Cα-OH of HMCA were selected and engineered by an iterative saturation mutation approach based on variant W2 (Additional file 1: Fig. S8C, D), resulting in variant W4 (S101A/H351V/N378S/Q329N). The specific activity of variant W4 toward CHDA was 4.21 ± 0.38 U·g−1, which represented a 1.5-fold, 1.7-fold, 3.4-fold, 4.4-fold, and 14.5-fold increase compared with the corresponding activities of variants W3, W2, W1*, W1, and WT, respectively (Table 1). As a result, the yield of CHDA increased to 40.1% and the yield of HMCA decreased to 56.6% (the titer decreased to 2.83 mM) (Fig. 4).
The kinetic parameters of the variants were determined, which are listed in Tables 1 and 2. The KMCHDA (5.24 ± 0.28 mM) and KMHMCA (1.12 ± 0.19 mM) values of variant W4 were 5.1-fold and 13.7-fold lower than those of WT, respectively, which indicated that the affinity with HMCA and CHDM of W4 was significantly enhanced. The kcatCHDA (0.94 ± 0.043 min−1) and kcatHMCA (2.20 ± 0.43 min−1) values of variant W4 increased by 21.8-fold and 13.8-fold, respectively, which indicated that variant W4 display a higher activity of primary alcohol oxidation. Accordingly, the kcat/KMCHDA (0.18 mM−1·min−1) and kcat/KMHMCA (1.96 mM−1·min−1) values increased by 112.5-fold and 196-fold, respectively. Although the selectivity of CHDA did not show complete reversal, the yield of CHDA and the RCHDA/HMCA increased to 40.1% and 0.72, respectively (Fig. 4). To evaluate the industrial-scale application potential of variant W4, CHDA was synthesized at the 3 L scale. Under the optimum conditions [72 g·L−1 (0.5 M) CHDM, 30 g·L−1 whole cell catalyst (wet E. coli), 5% (v/v) DMSO, 0.1 g·L−1 catalase, 0.1 M air-saturated potassium phosphate buffer, pH 8.0, and 30 °C], the titer of CHDA reached up to 29.6 g·L−1 in 12 h, with 42.2% yield and 2.5 g·L–1·h–1 space–time yield (STY) (Additional file 1: Figs. S10 and S11).
Structural analysis and performance enhancement mechanism of variant W4
To elucidate the mechanisms of the improved catalytic efficiency of the variant W4, the structure alignments of the WT-CHDM and W4-CHDM complex were elucidated, demonstrating that the structures are very similar with only slight differences in loop 100–103, loop 329–332, and the β-sheet, which is below the 4-Cα-OH of CHDM. The S101A mutation is located in loop 100–103 and is close to the catalytic center. The mutations H351V and N378S lie on the β-sheet, which is below the 4-Cα-OH of CHDM. In contrast, the mutation Q329N is located on loop 329–332, which is far away from the catalytic center (Fig. 5A, D). Therefore, interaction analysis was performed to better understand the loop swing. In the WT-CHDM, steric interactions occur between H351 and CHDM, and this interaction is weakened when mutated to the nonpolar residue V351, due to an increase in the distance between the side chain of V351 and the C atom of CHDM to 4.2 Å, which causes a dent in the steric effect (Fig. 5C). Moreover, the mutations N378S and H351V showed synergism, and further weakened the interactions between 4-Cα-OH of CHDM and the surrounding residues (Fig. 4). The residue S101 interacts with 1-Cα-OH of CHDM, and the W331 side chain forms an edge-to-face interaction with 4-Cα-OH of CHDM; thus, with the mutation of S101 to A101 and the mutation of Q329 to N329, the movement of loop 100–103 and loop 329–332 toward CHDM were restricted, and the distance between A101 and W331 with 1-Cα-OH and 4-Cα-OH of CHDM increased from 5.4 Å to 5.7 Å and from 2.6 Å to 4.3 Å, respectively (Fig. 5B, D and E). The root mean square fluctuation (RMSF) values of the residues were determined for both the WT and variant W4 using trajectory analysis, which revealed that the RMSF values of loop 100–103 and loop 329–332 of W4 were lower than those of WT (Fig. 5F). This result was consistent with the higher stability of loop 100–103 and loop 329–332. Therefore, the weakened flexibility of loop 100–103 and loop 329–332 in the variant W4 may contribute to restricting the inward loop swing, which prevents A101 and W331 from moving closer to 1-Cα-OH and 4-Cα-OH of CHDM and provides an extra space for conformation adjustment. This phenomenon was also observed in the WT-HMCA and W4-HMCA complex (Additional file 1: Fig. S12).
Therefore, the catalytically active conformation of CHDM and HMCA changed substantially in the variant. In the conformation of the W4-CHDM complex, the angle θN(10)-N(5)-(1-Cα-OH) decreased from 124.1° to 90.5°, thereby approaching 90° (Additional file 1: Fig. S13), in turn leading the D(1-Cα-H)-N(5)FAD and D(4-Cα-H)-N(5)FAD to decrease to 3.5 Å and 7.2 Å, respectively (Fig. 6A, B). The decreased distance in W4-CHDM is beneficial for the hydride (H−) transfer and the energy barrier of this step was decreased to 6.2 kcal·mol−1 (Fig. 3B). Moreover, the distance of the nucleophilic attack [D(1-Cα-OH)-Nε2H466 and D(4-Cα-OH)-Nε2H466] decreased to 2.9 Å and 8.4 Å, which facilitated the nucleophilic attack from H466 to 1-Cα-OH of CHDM, and consequently increased the catalytic efficiency of CHDM (Additional file 1: Fig. S14). In the conformation of the W4-HMCA complex, 4-Cα-OH of HMCA rotated to become buried in the active center, while 1-CHO of HMCA was anchored to the outside of the binding pocket, and these changes led the D(4-Cα-H)-N(5)FAD to decrease to 4.7 Å, while the D(4-Cα-OH)-Nε2H466 changed to 3.7 Å (Fig. 6C, D). The favorable conformation enhanced more HMCA to convert to CHDA, and the energy barrier of the hydride (H−) transfer was decreased to 9.2 kcal·mol−1, thereby facilitating the oxidization of 4-Cα-OH for the synthesis of CHDA, while simultaneously reducing HMCA accumulation in the bioconversion broth (Fig. 3B). In summary, the above-mentioned changes in the catalytically active conformation of CHDM and HMCA were beneficial in forming a lower energy barrier, leading to a higher transfer efficiency of hydride (H−) and more efficient primary alcohol oxidation by variant W4 to produce CHDA (29.6 g·L–1, 42.2% yield, STY 2.5 g·L–1·h–1).
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