Peptide bridging for cofactor channeling in fusion enzyme lowers cofactor input by two orders of magnitude

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

Abstract

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

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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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60231-7

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

Figures/Tables 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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