Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different

Abstract

The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.

FIG. 1

Comparison of the reactions catalyzed by melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 (A) and AtzA from Pseudomonas sp. strain ADP (B).

FIG. 2

Western blotting of an SDS-PAGE protein gel using antibodies against the AtzA protein. Lanes from left to right: 1, E. coli(pMD4) positive control for AtzA; 2, prestained protein standards; 3, Pseudomonas sp. strain ADP positive control for AtzA; 4, Pseudomonas sp. strain NRRL B-12228; 5, E. coli (TrzA) negative control; 6, Pseudomonas sp. strain NRRL B-12227; and 7, R. corallinus sp. strain NRRL B-15444R (TrzA).

FIG. 3

Amino acid sequences of AtzA and melamine deaminase (TriA) (GenBank accession no. AF312304). Boxes denote amino acid residues that differ between the two sequences.

FIG. 4

Time course of melamine and ammeline deamination by cell extracts prepared from the TriA clone E. coli(pJS3). Error bars represent the standard error of the mean; n = 2.