Directed evolution and characterization of atrazine chlorohydrolase variants with enhanced activity.
Atrazine chlorohydrolase (AtzA, EC 3.8.1.8) has attracted widespread interests as it catalyzes conversion of toxic atrazine to nontoxic hydroxyatrazine and can be used in the biodegradation of atrazine. To facilitate this application, a Haematococcus pluvialis-based method was applied to screen AtzA variants from a random mutagenesis library. Eight variants with enhanced enzyme activity were obtained. They showed 2.7- to 5.0-fold increase in specific activity compared with the wild type. Sequencing revealed that the two most active variants contained single substitution at Val12 and Leu395, respectively, while several improved variants contained substitutions at the four sites of Met315, His399, Asn429, and Val466 simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. Kinetic analysis showed that five variants decreased the Km value 0.6- to 0.9-fold, whereas all the variants increased the catalytic efficiency (kcat/Km value) 2.5- to 4.1-fold compared to the wild type. The modeled three-dimensional structure showed that AtzA is comprised of a typical (β/α)8 domain of the amidohydrolase superfamily and a dual β-sheet domain. An iron ion and five ligand-binding residues are located in the β-barrel core of the (β/α)8 domain. Some substituted residues are involved in hydrogen bond formation in the (β/α)8-neighboring β-sheet.
Laboratory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China. xiwenchen@nankai.edu.cn.
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D001280:Atrazine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology
D007527:Isoenzymes; D006867:Hydrolases; C100894:atrazine chlorohydrolase; D001280:Atrazine