title: 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.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, the University of Tokyo, Hongo, Tokyo, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Haga
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Hirakawa
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000595:Amino Acid Sequence; D019475:Camphor 5-Monooxygenase / Q000737:chemistry; D045304:Cytochromes c / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D005288:Ferredoxins; D009097:Multienzyme Complexes; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; C011083:polyproline; C100137:putidaredoxin; D045304:Cytochromes c; D019475:Camphor 5-Monooxygenase
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