title: Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate.
abstract: Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
authors.affiliation: Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Pavlova
authors.forename: Martin
authors.initials: M
authors.lastname: Klvana
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupkova
authors.forename: Pavel
authors.initials: P
authors.lastname: Banas
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Wade
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Jiri
authors.initials: J
authors.lastname: Damborsky
mesh_terms: D001665:Binding Sites; D001673:Biodegradation, Environmental; D002942:Circular Dichroism; D003198:Computer Simulation; D019020:Directed Molecular Evolution; D004785:Environmental Pollutants / Q000737:chemistry*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011407:Propane / Q000031:analogs & derivatives* / Q000737:chemistry; D015202:Protein Engineering; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D004785:Environmental Pollutants; C009536:1,2,3-trichloropropane; D006867:Hydrolases; C046137:haloalkane dehalogenase; D011407:Propane
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