title: Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.
abstract: Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.
authors.affiliation: Department of Chemical Engineering, University of Virginia, VA 22904, United States.
authors.forename: Sung In
authors.initials: SI
authors.lastname: Lim
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
authors.forename: Byungseop
authors.initials: B
authors.lastname: Yang
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
authors.forename: Younghan
authors.initials: Y
authors.lastname: Jung
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
authors.forename: Jaehyun
authors.initials: J
authors.lastname: Cha
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
authors.forename: Jinhwan
authors.initials: J
authors.lastname: Cho
authors.affiliation: School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.|Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwangju, 61186, Republic of Korea.
authors.forename: Eun-Sil
authors.initials: ES
authors.lastname: Choi
authors.affiliation: School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea.
authors.forename: Yong Hwan
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Department of Chemical Engineering, University of Virginia, VA 22904, United States.|School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry; D005560:Formate Dehydrogenases / Q000737:chemistry; D007700:Kinetics; D008353:Mannitol / Q000737:chemistry; D008354:Mannitol Dehydrogenases / Q000737:chemistry; D063747:Methanocaldococcus / Q000201:enzymology; D009097:Multienzyme Complexes / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D011336:Probability; D011551:Pseudomonas fluorescens / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013855:Thiobacillus / Q000201:enzymology
chemical_list: D009097:Multienzyme Complexes; D008353:Mannitol; D004247:DNA; D008354:Mannitol Dehydrogenases; D005560:Formate Dehydrogenases; D010100:Oxygen