title: Catalytic improvement and evolution of atrazine chlorohydrolase.
abstract: The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its k(cat)/K(m) value compared with that of the wild-type enzyme. The reduction in K(m) observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.
authors.affiliation: CSIRO Entomology, G.P.O. Box 1700, Canberra, ACT 2601, Australia. colin.scott@csiro.au
authors.forename: Colin
authors.initials: C
authors.lastname: Scott
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
authors.forename: Chris W
authors.initials: CW
authors.lastname: Coppin
authors.forename: Roslyn G
authors.initials: RG
authors.lastname: Mourant
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Hilton
authors.forename: Tara D
authors.initials: TD
authors.lastname: Sutherland
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001280:Atrazine / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D020125:Mutation, Missense; D017354:Point Mutation; D016415:Sequence Alignment
chemical_list: D006867:Hydrolases; C100894:atrazine chlorohydrolase; D001280:Atrazine