DOCSLIB.ORG

Atrazine Chlorohydrolase from Pseudomonas Sp

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Atrazine Chlorohydrolase from Pseudomonas Sp JOURNAL OF BACTERIOLOGY, Aug. 1996, p. 4894–4900 Vol. 178, No. 16 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology Atrazine Chlorohydrolase from Pseudomonas sp. Strain ADP: Gene Sequence, Enzyme Purification, and Protein Characterization† 1,2 2,3,4 1,2,4 MERVYN L. DE SOUZA, MICHAEL J. SADOWSKY, AND LAWRENCE P. WACKETT * Department of Biochemistry and Biological Processes Technology Institute,1 Department of Soil, Water, and Climate,3 Department of Microbiology,4 and Center for Biodegradation Research and Informatics,2 University of Minnesota, St. Paul, Minnesota 55108 Received 26 April 1996/Accepted 30 May 1996 Pseudomonas sp. strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine. The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M. L. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373–3378, 1995). In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine. The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chroma- tography. The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. The puri- 18 18 fied enzyme in H2 O yielded [ O]hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethyl- atrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine. Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater. The s-triazine ring is found as a constituent of herbicides, zines with one alkylamino group, such as desethylsimazine and dyes, and polymers. The s-triazine herbicides include simazine, desethylatrazine are metabolized by Rhodococcus corallinus terbutylazine, and atrazine [2-chloro-4-(ethylamino)-6-(isopro- NRRL B-15444R (8) via a hydrolytic enzyme that catalyzes pylamino)-1,3,5-triazine)]. The latter is the most environmen- both dechlorination and deamination reactions (28). The gene tally prevalent, being used for the control of broadleaf and encoding this hydrolase, trzA, was recently cloned and se- grassy weeds in major crops like corn, sorghum, and sugarcane. quenced (36). However, both Pseudomonas sp. strain NRRLB Atrazine is relatively persistent in soils (34). Atrazine occasion- 12227 and R. corallinus NRRL B-15444R were incapable of ally exceeds the U.S. Environmental Protection Agency maxi- metabolizing atrazine. Recently, bacteria that metabolize mum contaminant level of 3 ppb in groundwater and surface atrazine have been isolated (29, 32, 35, 36), but little informa- water (1, 5, 6, 14, 21, 22, 23, 30, 31). tion is available about the relevant metabolites, genes, and The metabolism of s-triazine compounds by pure bacterial enzymes. cultures has been studied (3, 4, 8–10, 15, 17, 20, 25–27, 32, 41, We previously reported the isolation of a pure bacterial 42), but most isolates failed to metabolize atrazine (9, 15). In culture, identified as Pseudomonas sp. strain ADP, which de- general, less-substituted s-triazine ring compounds are more graded relatively high concentrations of atrazine (.1,000 ppm) readily metabolized than their heavily substituted counter- under growth and nongrowth conditions (25). Pseudomonas sp. parts. As a result, information about the microbial genetics and strain ADP uses atrazine as the sole source of nitrogen for enzymology of s-triazine compounds has largely been obtained growth and liberates the ring carbon atoms as carbon dioxide. with compounds other than atrazine. For example, ammeline Recently, we reported the cloning, characterization, and ex- (2-hydroxy-4,6-diamino-s-triazine), which is not alkylated on pression of a DNA fragment from strain ADP that confers the ring-substituted amino groups, is metabolized by Pseudo- atrazine dechlorination ability on Escherichia coli DH5a (11). monas sp. strain NRRL B 12227 (12, 13). Its metabolism pro- The data indicate that hydroxyatrazine was the first interme- ceeds to cyanuric acid via two hydrolytic deamination reactions diate in the metabolism of atrazine by Pseudomonas sp. strain that are encoded by the genes trzB and trzC. Diamino-s-tria- ADP. The present study describes the sequence of the gene en- coding the atrazine dechlorination activity, designated atzA, * Corresponding author. Mailing address: Department of Biochem- istry, University of Minnesota, 140 Gortner Laboratories, 1479 Gort- and the purification of the corresponding enzyme (AtzA). ner Ave., St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625- Atrazine chlorohydrolase was characterized with respect to 5780. Electronic mail address: [email protected]. physical and catalytic properties. This is the first description of † Manuscript number 22,472 in the University of Minnesota Agri- a purified bacterial enzyme capable of catalyzing atrazine de- cultural Experiment Station series. chlorination. 4894 VOL. 178, 1996 CHARACTERIZATION OF ATRAZINE CHLOROHYDROLASE 4895 MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. Plasmid pMD4 (11), con- taining a 1.9-kb AvaI fragment in pACYC184 (7), was used in all studies. E. coli DH5a(pMD4) was used for the purification of atrazine chlorohydrolase. The culture was grown in Luria-Bertani LB medium (33), supplemented with 25 mg of chloramphenicol per ml, at 378C with shaking. DNA sequencing. Plasmid DNA was isolated as described previously (33). The nucleotide sequence of the approximately 1.9-kb AvaI DNA fragment, cloned in pMD4, was determined from both strands by using a PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer Corp., Norwalk, Conn.) and an ABI model 373A DNA sequencer (Applied Biosystems, Foster City, Calif.). Nucleotide sequence was determined initially by subcloning and subsequently by using primers designed with sequence information obtained from subcloned DNA fragments. The GCG sequence analysis software package (Genetics Computer Group, Inc., Madison, Wis.) was used for all DNA and protein sequence comparisons. DNA and protein sequences were compared with entries in Genbank, EMBL, PIR, and SwissProt sequence databases. Analytical methods. (i) Plate assays. Atrazine or hydroxyatrazine was incor- porated in solid LB (33) or minimal medium (25) at a final concentration of 500 mg/ml to produce an opaque suspension of small particles in the clear agar. The degradation of atrazine or hydroxyatrazine by wild-type and recombinant bacte- ria was indicated by a zone of clearing around atrazine-metabolizing colonies (11). (ii) HPLC analysis. High-performance liquid chromatography (HPLC) anal- ysis was performed by using a Hewlett-Packard HP 1090 Liquid Chromatograph system equipped with a photodiode array detector and interfaced to an HP 79994A Chemstation. Atrazine and its metabolites were resolved by using an analytical C18 reverse-phase NovaPak HPLC column (150 by 3.9 mm; Waters) 4-mm spherical packing; and an acetonitrile (ACN) gradient, in water, at a flow rate of 1.0 ml min21. Linear gradients were used as follows: 0 to 6 min, 10 to 25% ACN; 6 to 21 min, 25 to 65% ACN; 21 to 23 min, 65 to 100% ACN; and 23 to 25 min, 100% ACN. Spectral data of the column eluent were acquired between 200 and 400 nm (12-nm bandwidth per channel), with a sampling frequency of FIG. 1. Nucleotide sequence of atzA. The complete nucleotide sequence of 640 ms. Spectra were referenced against a signal at 550 nm and compared with the approximately 1.9-kb AvaI DNA fragment, cloned in pMD4, was determined those obtained by using authentic samples of atrazine and hydroxyatrazine. on both strands by the primer walking method and PCR. The atzA ORF is 18 16 (iii) MS analysis. H2 O (95%) and H2 O were purchased from Sigma Chem- indicated by the arrow, and a potential Pseudomonas ribosome binding site is ical Company (St. Louis, Mo.). For the labeling experiment, 50 ml of authentic underlined. The start and stop codons are underlined twice. atrazine or hydroxyatrazine at a concentration of 100 mg/ml in 25 mM MOPS buffer (pH 6.9) was added to each vial in two sets of glass vials. The samples were dried-down under vacuum and crimp sealed. The headspaces in the vials were repeatedly evacuated and flushed and finally filled with argon. The contents of The dialyzed protein fraction was loaded onto a Bio-Scale Q20 Anion Ex- 18 16 each vial was resuspended in 50 ml of either H2 OorH2 O by using a gas-tight change column (15 by 118 mm) (Bio-Rad Laboratories, Hercules, Calif.). The Hamilton syringe and allowed to equilibrate at room temperature for 60 min. column was washed with 25 mM MOPS buffer (pH 6.9), and the protein was The experiment was initiated by adding purified atrazine chlorohydrolase (AtzA) eluted using a 400-ml linear gradient of 0.0 to 0.5 M KCl at a flow rate of 2 to each vial. Reaction vials were shaken for 30 s and incubated at room temper- ml/min. Protein eluting from the column was monitored at 280 nm by using a ature for 5 h, and the reaction mixture was dried under vacuum.
Load more

Recommended publications
  • Biodegradation of Atrazine by Atrazine Chlorohydrolase: Characterization of Mutant Enzyme and Immobilization System for Water Purification
    Biodegradation of Atrazine by Atrazine Chlorohydrolase: Characterization of Mutant Enzyme and Immobilization System for Water Purification. A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Amit Aggarwal IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Lawrence P. Wackett & Ping Wang JANUARY 2012 © Amit Aggarwal 2011 Acknowledgement I want to thank my advisor, Dr. Larry Wackett, for the opportunity to work in his laboratory, his support and patience, and for allowing me to work independently and always being there to guide me. I would like to thank my mentor Dr. Jennifer Seffernick, for training me on different techniques in laboratory, and patiently helping me out throughout my association with the lab and teaching me good laboratory practices for research. Thank you so much for taking me under your wing. You are the best mentor I ever had. I would also like to thank Dr. Alptekin Aksan and Dr. Mike Sadowsky for guiding us throughout the immobilization of atrazine chlorohydrolase project. Dr. Alptekin Aksan guided us in development of a robust and efficient immobilization system and Dr. Mike Sadowsky provided us with valuable direction about making the immobilization system safe to be used in water purifications systems. I would like to thank my administrative adviser Dr. Ping Wang for always being supportive of my work and helping me out with planning my degree and ensuring that I meet all requirements in time. I am thankful to each of my committee members, Dr. Larry Wackett, Dr. Ping Wang and Dr. Jonathan Schilling for their time, for reading my thesis and for all the helpful suggestions.
    [Show full text]
  • Atrazine Mineralization Potential and Catabolic Gene Detection In
    ATRAZINE MINERALIZATION POTENTIAL AND CATABOLIC GENE DETECTION IN AGRICULTURAL AND WETLAND SITES DISSERTATION Presented in Partial Fulfillment of the Requirements of the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kristen Lynn Anderson, B. S. * * * * * The Ohio State University 2003 Dissertation Committee: Approved by Dr. Olli H. Tuovinen, Advisor Dr. Michael J. Boehm _____________________________ Dr. Mark Morrison Advisor Dr. Samuel J. Traina Department of Microbiology ABSTRACT Atrazine (2-chloro-4-ethylamine-6-isopropylamino-1,3,5 triazine) is a commonly applied herbicide in corn fields. Although the fate of atrazine in agricultural systems has been well studied, the environmental fate of atrazine in wetland systems is less well characterized. The majority of research in this area has focused on aerobic mineralization of atrazine, although anaerobic conditions are commonly found in wetland sediments and bulk soils associated with agricultural fields. The hypothesis for this work was that atrazine would be actively mineralized in agricultural and wetland sites. It was further hypothesized that active mineralization in soils could be correlated with the presence of selected genes involved in atrazine metabolism. Soil, sediment, and water samples were obtained from three sites in Ohio. Atrazine mineralization was investigated under aerobic and anaerobic conditions in these samples using a biometer system in 14 which CO2 evolution was correlated with atrazine mineralization. All samples mineralized atrazine under aerobic conditions. Under anaerobic conditions, some external electron acceptor amendments inhibited mineralization, while others enhanced it. The effect on mineralization varied with the sample and season. Attempts were made to amplify some of the genes involved in atrazine mineralization.
    [Show full text]
  • Proteomic Analysis of the Response of Funnelifor Mismosseae/Medicago Sativa to Atrazine Stress Xin Sui1,2† ,Qiwu1,2†, Wei Chang1,2, Xiaoxu Fan1,2 and Fuqiang Song1,2*
    Sui et al. BMC Plant Biology (2018) 18:289 https://doi.org/10.1186/s12870-018-1492-1 RESEARCH ARTICLE Open Access Proteomic analysis of the response of Funnelifor mismosseae/Medicago sativa to atrazine stress Xin Sui1,2† ,QiWu1,2†, Wei Chang1,2, Xiaoxu Fan1,2 and Fuqiang Song1,2* Abstract Background: Arbuscular mycorrhizal (AM) fungi form symbiotic associations with host plants can protect host plants against diverse biotic and abiotic stresses, and promote biodegradation of various contaminants. However, the molecular mechanisms of how the arbuscular mycorrhizal fungi and host plant association on atrazine stress were still poorly understood. To better characterize how arbuscular mycorrhizal fungi and host plant interactions increase atrazine stress, we performed physiological and proteomic analysis of Funneliformis mosseae (mycorrhizal fungi) and Medicago sativa (alfalfa) association under atrazine stress. Results: The results showed that in the Arbuscular mycorrhizal, protective enzymes were up regulated and the malondialdehyde content increased relative to those of non-mycorrhizal M.sativa. We also examined the atrazine degradation rates within the nutrient solution, and a 44.43% reduction was observed with the mycorrhizal M.sativa, with 30.83% of the reduction attributed to F. mosseae. The accumulation content in root and stem of mycorrhizal M.sativa were obviously increased 11.89% and 16.33% than those of non- mycorrhizal M.sativa. The activity of PPO, POD, CAT and SOD in mycorrhizal M.sativa were obviously higher than non mycorrhizal M.sativa under atrazine stess. We identified differential root proteins using isobaric tags for relative and absolute quantization coupled with liquid chromatography–mass spectrometry, with 533 proteins identified (276 unregulated and 257 downregulated).
    [Show full text]
  • Studies on the Biodegradation of Atrazine
    Universidade de Lisboa Faculdade de Ciências Departamento de Biologia Vegetal STUDIES ON THE BIODEGRADATION OF ATRAZINE IN SOILS CONTAMINATED WITH A COMMERCIAL FORMULATION CONTAINING ATRAZINE AND S-METOLACHLOR: SCALE-UP OF A BIOREMEDIATION TOOL BASED ON PSEUDOMONAS SP. ADP AND EVALUATION OF ITS EFFICACY Ana Catarina da Silva Portinha e Costa Mestrado em Biologia Celular e Biotecnologia Setembro 2008 Universidade de Lisboa Faculdade de Ciências Departamento de Biologia Vegetal STUDIES ON THE BIODEGRADATION OF ATRAZINE IN SOILS CONTAMINATED WITH A COMMERCIAL FORMULATION CONTAINING ATRAZINE AND S-METOLACHLOR: SCALE-UP OF A BIOREMEDIATION TOOL BASED ON PSEUDOMONAS SP. ADP AND EVALUATION OF ITS EFFICACY Ana Catarina da Silva Portinha e Costa Mestrado em Biologia Celular e Biotecnologia Dissertação orientada por: Doutora Cristina Anjinho Viegas (Instituto Superior Técnico, Universidade Técnica de Lisboa) Doutora Maria Isabel Caçador (Faculdade de Ciências, Universidade de Lisboa) Setembro 2008 ABSTRACT Atrazine has been used worldwide since 1952 and is frequently detected above the levels established by regulatory authorities in consumption waters. Therefore, and because of its ecotoxicological properties, its use has been forbidden in most European countries, including Portugal. However, atrazine is still used in many countries worldwide. The main purpose of the present work was to examine the efficacy of the atrazine- degrading bacteria Pseudomonas sp. ADP ( P. ADP) as bioaugmentation agent in soils contaminated with high doses (~20x and ~50xRD; RD – Recommended dose) of the commercial formulation, Primextra S-Gold, that contains atrazine, and also S-metolachlor and benoxacor as main active ingredients. It was also tested the effect of combining bioaugmentation and biostimulation using soil amendment with trisodium citrate in open soil microcosms, with the purpose of scaling-up this bioremediation tool.
    [Show full text]
  • The Influence of Vegetation, Microbial Inoculation, and Aging of Pesticide
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2001 The influence of vegetation, microbial inoculation, and aging of pesticide residues on the degradation of atrazine and metolachlor in soils Shaohan Zhao Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, Environmental Sciences Commons, and the Soil Science Commons Recommended Citation Zhao, Shaohan, "The influence of vegetation, microbial inoculation, and aging of pesticide residues on the degradation of atrazine and metolachlor in soils " (2001). Retrospective Theses and Dissertations. 693. https://lib.dr.iastate.edu/rtd/693 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, sbme thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.
    [Show full text]
  • New Aspects on Atrazine Biodegradation
    487 Vol.53, n. 2: pp. 487-496, March-April 2010 BRAZILIAN ARCHIVES OF ISSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL New Aspects on Atrazine Biodegradation Luciane Sene 1*, Attilio Converti 2, Geslaine Aparecida Ribeiro Secchi 1 and Rita de Cássia Garcia Simão 1 1Centro de Ciências Médicas e Farmacêuticas; Universidade Estadual do Oeste do Paraná; Rua Universitária 2069; Cascavel - PR - Brasil. 2Facoltà di Ingegneria; Dipartimento di Ingegneria Chimica e di Processo; Università degli Studi di Genova; Via dell’Opera Pia 1; I-16145; Genoa - Italy ABSTRACT The world practice of using agrochemicals for long periods, in an indiscriminated and abusive way, has been a concern of the authorities involved in public health and sustainability of the natural resources, as a consequence of environmental contamination. Agrochemicals refer to a broad range of insecticides, fungicides and herbicides, and among them stands out atrazine, a herbicide intensively used in sugarcane, corn and sorghum cultures, among others. Researches have demonstrated that atrazine has toxic effects in algae, aquatic plants, aquatic insects, fishes and mammals. Due to the toxicity and persistence of atrazine in the environment, the search of microbial strains capable of degrading it is fundamental to the development of bioremediation processes, as corrective tools to solve the current problems of the irrational use of agrochemicals. This review relates the main microbial aspects and research on atrazine degradation by isolated microbial species and microbial consortia, as well as approaches on the development of techniques for microbial removal of atrazine in natural environments. Key words : atrazine, biodegradation, bacteria, fungi, microbial consortium INTRODUCTION thus, inhibiting the photosynthetic electron transport.
    [Show full text]
  • Expression of Anti-Atrazine Scfv and Atrazine Chlorohydrolase Trzn in Planta for Potential Phytoremediation of Atrazine Contamination
    Expression of Anti-Atrazine scFv and Atrazine Chlorohydrolase TrzN in planta for Potential Phytoremediation of Atrazine Contamination By Siripen Leelachao A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Degree of Doctor of Philosophy In Environmental Biology and Toxicology Guelph, Ontario, Canada © Siripen Leelachao, April, 2015 ABSTRACT EXPRESSION OF ANTI-ATRAZINE SCFV AND ATRAZINE CHLOROHYDROLASE TRZN IN PLANTA FOR POTENTIAL PHYTOREMEDIATION OF ATRAZINE CONTAMINATION Siripen Leelachao Advisors: University of Guelph, 2014 Professor J. Christopher Hall Professor Keith R. Solomon Atrazine is extensively used in agricultural production to improve crop yield throughout North America. Due to contamination of surface water with atrazine, which is an environmental concern, tools for mitigating such contamination are needed. In this study, the potential use of genetically modified plants for remediation of atrazine contamination was investigated. Plants were genetically modified to express proteins of interest using Agrobacterium-mediated transformation. First, an anti-atrazine single chain variable fragment (scFv) was expressed in Lemna minor. In a hydroponic system, transgenic L. minor expressing the scFv showed higher tolerance to atrazine than wild- type plants. Absorption of 14C-atrazine in transgenic L. minor was greater than wild-type L. minor. Metabolites of atrazine were not found in transgenic L. minor. Second, L. minor was transformed to express atrazine chlorohydrolase (TrzN), but the protein could not be detected. Resistance to atrazine was found in the transgenic plants at low concentrations. Finally, the gene of atrazine chlorohydrolase TrzN was tested in Nicotiana Benthamiana by agroinfiltration. Transient Expression of atrazine ii chlorohydrolase TrzN and tolerance to atrazine in transgenic N.
    [Show full text]
  • The Ever-Expanding Limits of Enzyme Catalysis and Biodegradation: Polyaromatic, Polychlorinated, Polyfluorinated, and Polymeric Compounds
    Biochemical Journal (2020) 477 2875–2891 https://doi.org/10.1042/BCJ20190720 Review Article The ever-expanding limits of enzyme catalysis and biodegradation: polyaromatic, polychlorinated, polyfluorinated, and polymeric compounds Lawrence P. Wackett1,2,3 and Serina L. Robinson2,3 1 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Twin Cities, Minneapolis, MN 55108, U.S.A.; Biotechnology Institute, University of Downloaded from http://portlandpress.com/biochemj/article-pdf/477/15/2875/890352/bcj-2019-0720c.pdf by guest on 02 October 2021 Minnesota, Twin Cities, Minneapolis, MN 55108, U.S.A.; 3Program in Microbiology, Immunology and Cancer Biology, University of Minnesota, Twin Cities, Minneapolis, MN 55108, U.S.A. Correspondence: Lawrence P. Wackett ([email protected]) Biodegradation is simply the metabolism of anthropogenic, or otherwise unwanted, che- micals in our environment, typically by microorganisms. The metabolism of compounds commonly found in living things is limited to several thousand metabolites whereas ∼100 million chemical substances have been devised by chemical synthesis, and ∼100 000 are used commercially. Since most of those compounds are not natively found in living things, and some are toxic or carcinogenic, the question arises as to whether there is some organism somewhere with the enzymes that can biodegrade them. Repeatedly, anthropogenic chemicals have been denoted ‘non-biodegradable,’ only to find they are reactive with one or more enzyme(s). Enzyme reactivity has been organized into categor- ies of functional group transformations. The discovery of new functional group transfor- mations has continually expanded our knowledge of enzymes and biodegradation. This expansion of new-chemical biodegradation is driven by the evolution and spread of newly evolved enzymes.
    [Show full text]
  • Enhanced Atrazine Degradation: Evidence for Reduced Residual Weed Control and a Method for Identifying Adapted Soils and Predicting Herbicide Persistence L
    Weed Science 2009 57:427–434 Enhanced Atrazine Degradation: Evidence for Reduced Residual Weed Control and a Method for Identifying Adapted Soils and Predicting Herbicide Persistence L. Jason Krutz, Ian C. Burke, Krishna N. Reddy, Robert M. Zablotowicz, and Andrew J. Price* Soilborne bacteria with novel metabolic abilities have been linked with enhanced atrazine degradation and complaints of reduced residual weed control in soils with an s-triazine use history. However, no field study has verified that enhanced degradation reduces atrazine’s residual weed control. The objectives of this study were to (1) compare atrazine persistence and prickly sida density in s-triazine-adapted and nonadapted field sites at two planting dates; (2) utilize original and published data to construct a diagnostic test for identifying s-triazine-adapted soils; and (3) develop and validate an s- triazine persistence model based on data generated from the diagnostic test, i.e., mineralization of ring-labeled 14C-s- triazine. Atrazine half-life values in s-triazine-adapted soil were at least 1.4-fold lower than nonadapted soil and 5-fold lower than historic estimates (60 d). At both planting dates atrazine reduced prickly sida density in the nonadapted soils (P # 0.0091). Conversely, in the s-triazine-adapted soil, prickly sida density was not different between no atrazine PRE and atrazine PRE at the March 15 planting date (P 5 0.1397). A lack of significance in this contrast signifies that enhanced degradation can reduce atrazine’s residual control of sensitive weed species. Analyses of published data indicate that cumulative mineralization in excess of 50% of C0 after 30 d of incubation is diagnostic for enhanced s-triazine degradation.
    [Show full text]
  • HHS Public Access Author Manuscript
    HHS Public Access Author manuscript Author Manuscript Author ManuscriptAngew Chem Author Manuscript Int Ed Engl Author Manuscript . Author manuscript; available in PMC 2016 March 09. Published in final edited form as: Angew Chem Int Ed Engl. 2015 March 9; 54(11): 3351–3367. doi:10.1002/anie.201409470. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution Dr. Hans Renata, Dr. Z. Jane Wang, and Prof. Dr. Frances H. Arnold Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd. MC 210-41, Pasadena, CA 91125 (USA) Frances H. Arnold: [email protected] Abstract High selectivities and exquisite control over reaction outcomes entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not in nature’s known repertoire. We will use this review to outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progressions have been recapitulated in the laboratory starting from extant enzymes. We then examine non-native enzyme activities that have been discovered and exploited for chemical synthesis, emphasizing reactions that do not have natural counterparts. The new functions have mechanistic parallels to the native reaction mechanisms that often manifest as catalytic promiscuity and the ability to convert from one function to the other with minimal mutation. We present examples of how non-natural activities have been improved by directed evolution, mimicking the process used by nature to create new catalysts.
    [Show full text]
  • Nikolina Udiković-Kolić, Colin Scott & Fabrice Martin-Laurent
    Evolution of atrazine-degrading capabilities in the environment Nikolina Udiković-Kolić, Colin Scott & Fabrice Martin-Laurent Applied Microbiology and Biotechnology ISSN 0175-7598 Volume 96 Number 5 Appl Microbiol Biotechnol (2012) 96:1175-1189 DOI 10.1007/s00253-012-4495-0 1 23 Your article is protected by copyright and all rights are held exclusively by Springer- Verlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Appl Microbiol Biotechnol (2012) 96:1175–1189 DOI 10.1007/s00253-012-4495-0 MINI-REVIEW Evolution of atrazine-degrading capabilities in the environment Nikolina Udiković-Kolić & Colin Scott & Fabrice Martin-Laurent Received: 17 August 2012 /Revised: 2 October 2012 /Accepted: 3 October 2012 /Published online: 18 October 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract Since their first introduction in the mid 1950s, man- Introduction made s-triazine herbicides such as atrazine have extensively been used in agriculture to control broadleaf weed growth in Increasing crop production in agriculture has been made different crops, and thus contributed to improving crop yield easier by the use of effective chemical weed control agents, and quality. Atrazine is the most widely used s-triazine herbi- such as the s-triazine herbicides.
    [Show full text]
  • Impact of Atrazine Exposure on the Microbial Community Structure in A
    Microbes Environ. Vol. 35, No. 2, 2020 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME19143 Impact of Atrazine Exposure on the Microbial Community Structure in a Brazilian Tropical Latosol Soil Ana Flavia Tonelli Fernandes1,2, Ping Wang2, Christopher Staley2, Jéssica Aparecida Silva Moretto1, Lucas Miguel Altarugio3, Sarah Chagas Campanharo1, Eliana Guedes Stehling1, and Michael Jay Sadowsky2,4* 1Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Café Avenue s/n, Ribeirão Preto, SP 14040–903, Brazil; 2Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, 55108 Saint Paul, MN 55108, USA; 3Department of Soil Science ESALQ, University of São Paulo, 11 Pádua Dias Avenue, Piracicaba, SP 13418–260, Brazil; and 4Department of Soil, Water, & Climate, 1991 Upper Buford Circle and Department of Plant and Microbial Biology, 1479 Gortner Avenue— University of Minnesota, Saint Paul, MN 55108, USA (Received November 8, 2019—Accepted February 17, 2020—Published online April 9, 2020) Atrazine is a triazine herbicide that is widely used to control broadleaf weeds. Its widespread use over the last 50 years has led to the potential contamination of soils, groundwater, rivers, and lakes. Its main route of complete degradation is via biological means, which is carried out by soil microbiota using a 6-step pathway. The aim of the present study was to investigate whether application of atrazine to soil changes the soil bacterial community. We used 16S rRNA gene sequencing and qPCR to elucidate the microbial community structure and assess the abundance of the atrazine degradation genes atzA, atzD, and trzN in a Brazilian soil.
    [Show full text]