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.

FIG. 1.

Alignment of the amino acid sequences of 1J6P and AtzA. The homology between AtzA and 1J6P is indicated. Conserved active-site- and substrate-binding pocket residues are in boldface.

FIG. 2.

Model of atrazine (center) docked in the active site of AtzA. The residues forming the hydrophobic base (Phe84, Trp87, and Leu88 [white]), the residues involved in coordination of the substrate (Glu246, Asn328, and Ser331 [purple]), the N-isopropyl binding pocket (Ala216, Thr217, Thr219, Ala220, and Asp250 [green]), and the N-ethyl binding pocket (Gln71, Gln96, Asn126, and Met155 [blue]) are shown. The residues that coordinate the active-site metal (His66, His68, His243, His276, and Asp327) have been omitted for clarity.

FIG. 3.

Distribution of the improvements (n-fold) in the specific activities of variants selected from the limited site saturation library. Specific activities were determined using a 23 μM initial concentration of atrazine and 34.6 nM enzyme.

FIG. 4.

(Top) Kinetic profiles for dechlorination of atrazine by wild-type AtzA (squares) and the AtzA 734 variant (triangles) as determined by colorimetric assay and by wild-type AtzA as determined by LC-MS (circles); (bottom) deamination of melamine by TriA as determined by LC-MS. Data varied by less than 10% in replicate experiments.

FIG. 5.

Model of atrazine docked in the active site of the wild-type AtzA N-isopropyl pocket (A) and the consensus N-isopropyl pocket variant of AtzA (B). Residues in the N-isopropyl pocket that were randomized are shown, demonstrating the closer contacts between the enzyme and substrate in the consensus variant.

FIG. 6.

Proposed catalytic mechanism for AtzA-mediated dechlorination of atrazine. The reaction proceeds by nucleophilic attack at C-4 of atrazine by the hydroxyl nucleophile (A), leading to the formation of the tetrahedral intermediate (B), followed by breakdown of the intermediate and departure of the chloride leaving group (C). Other residues that coordinate the active-site metal (His66, His68, His243, His276) have been omitted for clarity.