Characteristic Isotope Fractionation Patterns in <i>s</i>‑Triazine Degradation Have Their Origin in Multiple Protonation Options in the <i>s</i>‑Triazine Hydrolase TrzN

Abstract

<i>s</i>-Triazine herbicides (atrazine, ametryn) are groundwater contaminants which may undergo microbial hydrolysis. Previously, inverse nitrogen isotope effects in atrazine degradation by <i>Arthrobacter aurescens</i> TC1 (i) delivered highly characteristic (¹³C/¹²C, ¹⁵N/¹⁴N) fractionation trends for pathway identification and (ii) suggested that the <i>s</i>-triazine ring nitrogen was protonated in the enzyme <i>s</i>-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 <i>normal carbon isotope effects</i> (ε_carbon = −5.0‰ ± 0.2‰ (atrazine/TrzN), ε_carbon = −4.2‰ ± 0.5‰ (atrazine/TrzN-E241Q), ε_carbon = −2.4‰ ± 0.3‰ (ametryn/TrzN)) and <i>inverse nitrogen isotope effects</i> (ε_nitrogen = 2.5‰ ± 0.1‰ (atrazine/TrzN), ε_nitrogen = 2.1‰ ± 0.3‰ (atrazine/TrzN-E241Q), ε_nitrogen = 3.6‰ ± 0.4‰ (ametryn/TrzN)). Surprisingly, TrzN-E241Q therefore still activated substrates through protonation implicating another proton donor besides E241. Sulfur isotope effects were larger in enzymatic (ε_sulfur = −14.7‰ ± 1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (ε_sulfur = −0.2‰ ± 0.0‰, pH 1.75), indicating rate-determining <i>C–S</i> bond cleavage in TrzN. Our results highlight a robust inverse ¹⁵N/¹⁴N fractionation pattern for identifying microbial <i>s</i>-triazine hydrolysis in the environment caused by multiple protonation options in TrzN