Fine tuning of spatial arrangement of enzymes in a PCNA-mediated multienzyme complex using a rigid poly-L-proline linker

Abstract

Inspired by natural multienzyme complexes, many types of artificial multienzyme complexes have recently been constructed. We previously constructed a self-assembled complex of a bacterial cytochrome P450 and its ferredoxin and ferredoxin reductase partners using heterotrimerization of proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus. In this study, we inserted different peptide linkers between ferredoxin and the PCNA subunit, and examined the effect on activity of the self-assembled multienzyme complex. Although the activity was affected by the lengths of both the rigid poly-L-proline-rich linkers and the flexible Gly4-Ser repeating linkers, the poly-L-proline-rich linkers provided the greatest activity enhancement. The optimized poly-L-proline-rich linker enhanced the activity 1.9-fold compared with the GGGGSLVPRGSGGGGS linker used in the previously reported complex, while the Gly4-Ser repeating linkers, (G4S)n (n = 1-6), did not yield higher activity than the maximum activity by the optimized poly-L-proline linker. Both the rigidity/flexibility and length of the peptide linker were found to be important for enhancing the overall activity of the multienzyme complex.

Figure 1. Optimization of the PCNA2-PdX fusion protein linker in PUPPET.

(A) Model depicting the self-assembly of PCNA1-PdR, PCNA2-PdX and PCNA3-P450cam. (B) Schematic representation of 11 PCNA2-PdX fusion proteins (P₁-P₅ and G₁-G₆). The calculated molecular masses of the PCNA2-PdX linker variants are listed in the tables. MM, molecular mass.

Figure 2. SDS-PAGE analyses of PCNA2-PdX fusion proteins and PUPPETs.

(A) PCNA2-PdX fusion proteins: lane 1–5, PCNA2-G ₄S(P₅)_nG₄S-PdX (n = 1–5); lane 6–11, PCNA2-(G₄S)_n-PdX (n = 1–6) (B) PUPPETs: lane 1–5, PUPPET-P_n (n = 1–5); lane 6–11, PUPPET-G_n (n = 1–6). P1-PdR, P2-PdX and P3-P450 are PCNA1-PdR, PCNA2-PdX and PCNA3-P450cam, respectively.

Figure 3. UV–Vis spectra of PCNA2-G ₄S-PdX and PUPPET-G₁.

(A) UV–Vis spectrum of PCNA2-G ₄S-PdX. The peaks at 314, 412, and 460 nm, and the broad band in the long wavelength region, are specific to [2Fe-2S] cluster-containing proteins. UV–Vis spectra of PCNA2-(G₄S)_n-PdX (n = 2–6) and PCNA2-G ₄S(P₅)_nG₄S-PdX (n = 1–5) are shown in Figure S1. (B) UV–Vis spectra of PUPPET-G₁ (solid line), and a linear combination of the individual component protein spectra (broken line). PUPPET-G_n (n = 2–6) and PUPPET-P_n (n = 1–5) are shown in Figure S2.

Figure 4. Cytochrome c reduction activities of the PUPPET linker variants.

The activities of (A) PUPPET-P_n (n = 1–5) and (B) PUPPET-G_n (n = 1–6) were evaluated in a reaction mixture containing 4.5 nM enzymes and 20 µM cytochrome c. Error bars represent the standard errors of three replicates.

Figure 5. P450cam oxidation activities of the PUPPET linker variants.

The activities of (A) PUPPET-P_n (n = 1–5) and (B) PUPPET-G_n (n = 1–6) were evaluated in reaction mixtures containing 18 nM enzyme. The activity of the previously reported PUPPET, containing a GGGGSLVPRGSGGGGS linker (indicated as “O”), and the activity of an equimolar mixture of PdR, PdX and P450cam (indicated as “R”), were also evaluated under the same reaction conditions. Error bars represent the standard error of three replicates; *P < 0.05, ***P < 0.001 (Student’s t-test).

Figure 6. PUPPET concentration-dependent monooxygenase activities.

(A) Initial rates and (B) specific activities were plotted against the protein concentration of PUPPET-P₄ (open squares), PUPPET-G₅ (open triangles) and original PUPPET (closed circles). Each specific activity was normalized using the activity at 72 nM in Figure 6B. Error bars represent the standard error of three replicates.

Figure 7. Models for binding between PdX and P450cam in PUPPET.

(A) A docking model of P450cam and PdX. The docking program GRAMM-X [42] was used to generate the model from crystal structures of P450cam (PDB: 1DZ4) and PdX (PDB: 1XLP) according to a previous report [40]. (B, C) Spatial arrangement of P450cam and the PCNA ring when the PdX-binding site of P450cam faces (B) in the same direction as or (C) in a perpendicular direction to the PCNA ring. The distance between the C-termini of PCNA2 and PCNA3 was estimated from the crystal structure of the PCNA heterotrimer (PDB: 2NTI).