Characteristic isotope fractionation patterns in <em>s</em>-triazine degradation have their origin in multiple protonation options in the <em>s</em>-triazine hydrolase trzn.

s-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by Arthrobacter aurescens TC1 (i) delivered highly characteristic (13C/12C, 15N/14N) fractionation trends for pathway identification and (ii) suggested that the s-triazine ring nitrogen was protonated in the enzyme s-triazine hydrolase (TrzN) where (iii) TrzN crystal structure and mutagenesis indicated H+-transfer from the residue E241. This study tested the general validity of these conclusions for atrazine and ametryn with purified TrzN and a TrzN-E241Q site-directed mutant. TrzN-E241Q lacked activity with ametryn; otherwise, degradation consistently showed normal carbon isotope effects (&epsilon;carbon = -5.0&permil; &plusmn; 0.2&permil; (atrazine/TrzN), &epsilon;carbon = -4.2&permil; &plusmn; 0.5&permil; (atrazine/TrzN-E241Q), &epsilon;carbon = -2.4&permil; &plusmn; 0.3&permil; (ametryn/TrzN)) and inverse nitrogen isotope effects (&epsilon;nitrogen = 2.5&permil; &plusmn; 0.1&permil; (atrazine/TrzN), &epsilon;nitrogen = 2.1&permil; &plusmn; 0.3&permil; (atrazine/TrzN-E241Q), &epsilon;nitrogen = 3.6&permil; &plusmn; 0.4&permil; (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (&epsilon;sulfur = -14.7&permil; &plusmn; 1.0&permil;, ametryn/TrzN) than in acidic ametryn hydrolysis (&epsilon;sulfur = -0.2&permil; &plusmn; 0.0&permil;, pH 1.75), indicating rate-determining C-S bond cleavage in TrzN. Our results highlight a robust inverse 15N/14N fractionation pattern for identifying microbial s-triazine hydrolysis in the environment caused by multiple protonation options in TrzN

The enzymatic basis for pesticide bioremediation

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function