具有辅因子通道效应的肽桥融合酶降低辅因子用量两个数量级

doi:10.1016/S1872-2067(24)60231-7

摘要/Abstract

摘要：

氧化还原酶是最大的一类酶, 广泛应用于医药、食品和化工领域的生物催化反应. 约90%的氧化还原酶依赖烟酰胺腺嘌呤二核苷酸(磷酸) (NAD(P))作为辅因子, 通过电子传递完成催化反应. NAD(P)的稳定性差、成本高和用量大, 因此, 提高这类酶在生物催化中的辅因子利用效率具有重要意义. 酶融合是常用的辅因子再生策略, 但高用量和有限的辅因子回收性限制了催化过程的可持续性. 因此, 亟需开发降低NAD(P)的用量的方法, 以促进氧化还原酶催化过程的高效化.
本文提出了设计具有辅因子通道效应的肽桥融合氧化还原酶(苯丙酮单加氧酶和亚磷酸脱氢酶), 以通过辅因子通道效应降低融合酶(FuE)催化过程辅因子浓度的策略. 首先利用分子动力学模拟设计筛选对NADP具有高亲和性的氨基酸并构建连接肽, 通过实验验证, 获得了5种性能优于柔性肽(GGGGS)₂连接的融合酶. 其中, RRRQRRRARR肽桥连接的融合酶(FuE-R10)表现出最佳性能, 在NADP与酶的浓度比为0.1时, 催化转化率分别是混合游离酶和(GGGGS)₂融合酶的2.1倍和2.0倍. 酶催化分析表明, FuE-R10的NADP传输效率显著优于其他融合酶. 分子动力学模拟结果显示, 肽桥R10与NADP具有较高的解离能, 证明该融合酶通过肽桥建立了辅因子通道, 促进了NADP在两个酶活性位点之间的传输(减少了其向溶液中的自由扩散). 在多酶竞争实验中, FuE-R10有效抑制了NADPH的副氧化反应, 进一步验证了辅因子通道效应. 离子强度对级联反应的影响研究表明, 阳离子肽桥的辅因子静电通道效应促进了负电荷NADP在酶间的传输, 从而在低辅因子浓度下获得了较高的催化转化率. 缩短的肽桥RRRRR连接的融合酶(FuE-R5)可进一步增强通道效应, 表现为对副氧化反应的抑制作用增强和催化转化率提高. 与混合游离酶相比, FuE-R5在酯合成反应中可将NADPH浓度降低两个数量级(在1 μmol L^-1 NADPH条件下, 5 μmol L^-1 FuE-R5的转化率相当于5 μmol L^-1混合游离酶在150 μmol L^-1 NADPH下的转化率). 在相同的辅因子浓度下, FuE-R5的催化速率是混合酶的3倍.
综上, 本研究证明了辅因子通道肽桥策略可显著降低融合酶催化过程的辅因子浓度和提高催化反应速率, 为开发高效可持续级联生物催化过程提供了新型生物催化剂构建方法.

关键词: 肽桥, 融合酶, NADP(H)依赖性氧化还原酶, 辅因子通道, 辅因子再生

Abstract:

Biocatalysis with nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidoreductases faces a challenge in improving the efficiency of the costly cofactor utilization. Although enzyme fusion can offer cofactor regeneration, the high-volume input and limited cofactor recyclability still make the enzymatic processes unsustainable. Therefore, it is of great significance to reduce cofactor input in a fusion enzyme (FuE) system, but no successful practice has been reported. Herein, we design a decapeptide bridge, RRRQRRRARR (R10), with high affinity for NADPH to construct fusion oxidoreductases (phenylacetone monooxygenase and phosphite dehydrogenase) for ester synthesis and NADP recycling. The peptide bridge enables electrostatic cofactor channeling that transports NADPH/NADP⁺ across the peptide between the enzymes’ NADP-binding pockets, so the fusion enzyme (FuE-R10) presents 2.1-folds and 2.0-folds higher conversions than mixed free enzymes and a flexible linker (GGGGSGGGGS)-fused enzyme, respectively, at NADPH/FuE of 0.1. The fusion enzyme, FuE-R5, bridged by a half-shortened linker, is proved more effective in facilitating cofactor channeling; compared to the mixed free enzymes, FuE-R5 exhibits two orders of magnitude reduction of NADPH input in ester synthesis. The work has thus demonstrated the potential of the cofactor bridging strategy in the development of sustainable cofactor-dependent cascade biocatalysis.

Key words: Peptide bridge, Fusion enzyme, NADP(H)-dependent oxidoreductases, Cofactor channeling, Cofactor regeneration

郭泽辉, 孙彦. 具有辅因子通道效应的肽桥融合酶降低辅因子用量两个数量级[J]. 催化学报, 2025, 71: 390-403.

Zehui Guo, Yan Sun. Peptide bridging for cofactor channeling in fusion enzyme lowers cofactor input by two orders of magnitude[J]. Chinese Journal of Catalysis, 2025, 71: 390-403.

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链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(24)60231-7

https://www.cjcatal.com/CN/Y2025/V71/I4/390

图/表 6

Scheme 1. Schematic diagram illustrating the procedure for analysis of the interactions of NADPH and VPs by MD simulations (A), design of EPs based on the high-affinity VPs (B), validation of peptide linkers (EPs) for constructing fusion enzymes by MD simulations (C), and characterization of the peptide-bridged fusion enzyme for NADPH/NADP+ channeling by experiments and computations (D).

Fig. 1. Identification of high-affinity VPs for NADPH by MD simulations. (A) Schematic representation for the modeling of single-amino acid VPs, MD simulations of VPs/NADPH complexes, and analysis of the interactions between VPs and NADPH. (B,C) Radial distribution function of NADPH around VPs. (D) Portion function of NADPH around VPs. (E) Binding free energies between VPs and NADPH. (F) Portion function of NADPH around GS10 and EPs. (G) Binding free energies between GS10/EPs and NADPH. Data represent mean ± s.d. (n = 4).

Fig. 2. Investigation of NADP transportation effectiveness of EPs in FuEs by multi-enzyme catalytic reaction. (A) Relative conversions by MFEc and fusion enzymes at different NADPH/enzyme ratios (CN:CE), in which the relative conversion by MFEc was set as 100%. (B) NADP transportation effectiveness factor α for fusion enzymes at different NADPH concentration ratios (μmol L-1/μmol L-1). (C) Conversions by FuE-R10 and MFEa (the same single-enzyme activities) at different CN: CE (PAMO or FuE-R10 concentration) values. (D) NADP transportation effectiveness factor β for fusion enzymes at different CN:CE (PAMO or FuE concentration) values. * p < 0.05, ** p < 0.01, and *** p < 0.001. Data represent mean ± s.d. (n = 3). Enzyme concentrations were all 1 μmol L-1.

Fig. 3. Analysis of NADP channeling in the FuEs-catalyzed reaction. (A) Transition state areas (circled positions) identified by molecular docking simulations for the binding of NADPH to PAMO and for the binding of NADP+ to PTDH. (B) System configuration of NADPH and PAMO-peptide complex (complex 1) and NADP+ and peptide-PTDH complex (complex 2) when NADPH or NADP+ is released around the transition state (point b or e), respectively. a and d are the first and last residues of the peptide linker, and c and f are the NADPH-binding and NADP+-binding sites in PAMO and PTDH pockets, respectively. (C) Probabilities for the final states of NADPH and NADP+ (dissociated state, on peptide bridge, in enzyme’s NADP-binding pocket). (D) The dissociation energy barrier (?GPAMO and ?GPTDH) values for NADPH and NADP+ on complexes 1 and 2, respectively. (E) Schematic representation of the competitive side reaction experiment. (F) Relative H2O2 production in the presence of MFEa, FuE-GS10, and FuE-R10 in competitive side reaction experiment, in which the relative H2O2 production in the presence of MFEa was set as 100%. Enzyme concentrations were all 0.5 μmol L-1. * p < 0.05 and ** p < 0.01. Data represent mean ± s.d. (n = 3).

Fig. 4. Evidence of electrostatic NADP channeling. (A) Relative conversions by MFEa, FuE-GS10, and FuE-R10 at different buffer and/or NaCl concentrations. (B) PAMO activities of free PAMO and fusion enzymes. (C) PTDH activities of free PTDH and fusion enzymes. (D) Relative conversions by MFEc and fusion enzymes at different NADPH/enzyme ratios (CN:CE). (E) NTEF α for fusion enzymes at an NADPH concentration ratio of 0.1:1. (F) and (G) Conversions by MFEa and fusion enzymes at CN:CE (PAMO or FuE concentration) = 1.0 and 0.1. (H) NTEF β for fusion enzymes at different CN:CE (PAMO or FuE concentration) values. FuE concentrations were all 1 μmol L-1. (I) Relative H2O2 production in the presence of MFEa and FuE-R5 in competitive side reaction experiment, in which the relative H2O2 production in the presence of MFEa was set as 100%. Fusion enzymes were FuE-GS10, FuE-R5, FuE-R10, and FuE-R20. Enzyme concentrations were all 0.5 μmol L-1. * p < 0.05, ** p < 0.01, and *** p < 0.001. Data represent mean ± s.d. (n = 3).

Fig. 5. Ester synthesis catalyzed by MFEc and FuE-R5. (A) Conversions by MFEc and FuE-R5 at various NADPH concentrations and at different time periods. (B) Comparison of the 24-h conversions by MFEc at different NADPH concentrations and FuE-R5 at 1 μmol L-1 NADPH. Cascade catalytic reactions of MFEc and FuE-R5 were performed in Tris-HCl buffer (50 mmol L-1, pH = 7.5) containing 5 μmol L-1 enzyme, 5 mmol L-1 BiCO, 10 mmol L-1 phosphite (Na2PO3), and 10 μmol L-1 FAD at 24 °C and 150 rpm. Data represent mean ± s.d., n = 3.

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