title: Characteristic isotope fractionation patterns in s-triazine degradation have their origin in multiple protonation options in the s-triazine hydrolase TrzN.
abstract: 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 (εcarbon=-5.0‰±0.2‰ (atrazine/TrzN), εcarbon=-4.2‰±0.5‰ (atrazine/TrzN-E241Q), εcarbon=-2.4‰±0.3‰ (ametryn/TrzN)) and inverse nitrogen isotope effects (εnitrogen=2.5‰±0.1‰ (atrazine/TrzN), εnitrogen=2.1‰±0.3‰ (atrazine/TrzN-E241Q), εnitrogen=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 (εsulfur=-14.7‰±1.0‰, ametryn/TrzN) than in acidic ametryn hydrolysis (εsulfur=-0.2‰±0.0‰, 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.
authors: †Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Bavaria, Germany.
authors: Heide K V
authors: HK
authors: Schürner
authors: ‡Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States.
authors: Jennifer L
authors: JL
authors: Seffernick
authors: §Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors: Anna
authors: A
authors: Grzybkowska
authors: §Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors: Agnieszka
authors: A
authors: Dybala-Defratyka
authors: ‡Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota 55108, United States.
authors: Lawrence P
authors: LP
authors: Wackett
authors: †Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Bavaria, Germany.
authors: Martin
authors: M
authors: Elsner
mesh_terms: D001173:Arthrobacter / Q000235:genetics / Q000378:metabolism*; D001280:Atrazine / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002247:Carbon Isotopes / Q000378:metabolism*; D005591:Chemical Fractionation; D006540:Herbicides / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D006868:Hydrolysis; D009587:Nitrogen Isotopes / Q000378:metabolism; D014227:Triazines / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D006540:Herbicides; D009587:Nitrogen Isotopes; D014227:Triazines; D014874:Water Pollutants, Chemical; C100057:ametryne; D006867:Hydrolases; C100894:atrazine chlorohydrolase; D001280:Atrazine