Introduction of selective intersubunit disulfide bonds into self‐assembly protein scaffold to enhance an artificial multienzyme complex's activity

Abstract

In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self-assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring-shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co-localized on the PCNA ring by fusing them to the C-termini of the three PCNA subunits. However, an advantage of the specific non-covalent complex formation is inextricably associated with the disadvantage of its concentration-dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components.

Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker

“…First, two Sap I sites were inserted by PCR between the genes encoding PCNA2 and the Cys73Ser/Cys85Ser mutant of PdX in pHSG+PCNA2-PdX [17], using two primers, 5’-GGATCCGCTCTTCAGGAGGTGGTGGCTCTATGTCTAAAG-3’ (forward) and 5’-GAATTCGCTCTTCTACCGCCGTCCGCGC-3’ (reverse). The DNA fragment, containing two Sap I sites between PCNA2 and PdX, was amplified from the resulting plasmid by PCR using two primers, 5’-TATTTCCAGGGCCATATGATGAAAGCCAAAGTGATCG-3’ (forward) and 5’-ATGGCCCTGGAAATACAGG-3’ (reverse).…”

Phosphite‐driven Self‐sufficient Cytochrome P450

“…[13] PCNA2-PdX was expressed and purified as described previously, [29] except that BL21 Star (DE3) pLysS was used for expression. PCNA3-P450cam was expressed from pET15b + P3MC using T7 Express I q by inoculating cells in TB medium containing 1 mm 5-aminolevulinic acid hydrochloride and cultivating cells at 27 8C, and purified as described previously.…”

“…PCNA1-PdR was expressed by BL21 Star (DE3) pLysS transformed with pET28b + P1R, [29] and purified as described previously. [13] PCNA2-PdX was expressed and purified as described previously, [29] except that BL21 Star (DE3) pLysS was used for expression.…”

“…The gene encoding PCNA2-GGGGSLVPRGSGGGGS-PdX was amplified from a plasmid, pHSG + PCNA2 + PdX, [29] by PCR using two primers: The generated DNA fragment was inserted between the SapI and BamHI sites of pPTDHsap. The resulting plasmid, pPD2X, comprised the expression vector for PTDH-PCNA2-PdX.…”

“…A plasmid, pHSG + PCNA3-P450cam, [29] was used as a template for PCR to introduce the N106R mutation into PCNA3 using two primers:…”