ATZC IS A NEW MEMBER OF THE AMIDOHYDROLASE PROTEIN SUPERFAMILY AND ISHOMOLOGOUS TO OTHER ATRAZINE-METABOLIZING ENZYMES

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via t hree plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hy droxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deam idation, yielding N-isopropylammelide. In this study, the third gene i n the atrazine catholic pathway, atzC, was cloned from a Pseudomonas s p. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escheric hia coli. The atzC gene mas further delimited by functional analysis f ollowing transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-Av aI fragment, An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N- isopropylammelide stoichiometrically to cyanuric acid and N-isopropyla mine. The 2.0-kb DNA fragment was sequenced and found to contain a sin gle open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, resp ectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily, The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the f ive ligands to the catalytically important metal for the protein, Pair wise comparison of the 35 amino acids showed 61% sequence identity and 55% sequence similarity, AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deami nase, and phosphotriester hydrolase, Similar sequence comparisons of t he most highly conserved regions indicated that the AtzA and AtzB prot eins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.