title: Surface orientation control of site-specifically immobilized nitro-reductase (NfsB).
abstract: We demonstrate the control of enzyme orientation for enzymes chemically immobilized on surfaces. Nitro-reductase (NfsB) has the ability to reduce a broad range of nitro-containing compounds and has potential applications in a broad range of areas including the detection and decomposition of explosives. The enzyme was tethered through unique surface cysteine residues to a self-assembled monolayer (SAM) terminated with maleimide groups. One cysteine was introduced close to the active site (V424C), and the other, at a remote site (H360C). The surface-tethered NfsB variants were interrogated by a combination of surface-sensitive sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) to determine how the mode of attachment altered the enzyme's orientation. The activities of the two immobilized NfsB variants were measured and can be well correlated to the deduced orientations. The relationships among enzyme engineering, surface immobilization, enzyme orientation, and enzyme activity were revealed.
authors.affiliation: Department of Chemistry and ‡Chemical Biology Graduate Program, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Shen
authors.forename: McKenna
authors.initials: M
authors.lastname: Schroeder
authors.forename: Tadeusz L
authors.initials: TL
authors.lastname: Ogorzalek
authors.forename: Pei
authors.initials: P
authors.lastname: Yang
authors.forename: Fu-Gen
authors.initials: FG
authors.lastname: Wu
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
authors.forename: Zhan
authors.initials: Z
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D009601:Nitroreductases / Q000737:chemistry* / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; C116054:NfsB protein, E coli; D009601:Nitroreductases