Chapter4 The Rote of Plant and Microbial Hydrolytic Enzymesin PesticideMetabolism

Robert E. Hoaglandand Robert M. Zablotowicz

SouthernWeed ScienceResearch Unit, Agricultural Research Service,U.S. Departmentof Agriculture, Sloneville,MS 38776

Many pesticide moleculescontaining amide or carbamatebonds, or esterswith carbonyl, phosphoryl,and thionyl linkages,are subjectto enzymatic hydrolysis. Pesticide hydrolysis by esterases and amidases from plants and microorganlsms can serve as a deloxification or activation mechanism that can govern pesticide sel€ctivity or resistance, and initiate or determine the rate of pesticide biodegradationin the environment. Substratespecificity of esterases and amidases varies dramatically among species and biotypes of plants and microorganisms. The constitutive or induJible nature of these , as well as production of isozymes,is also important in the expressionof thesemechanisms' For example,'increasedaryl acylamidaseactivity has been reported as the mechanismof evolved resistanceto the hedicide in two Echinochloq weed species fiunglerice, E. colona (L.) Link; barnyardgrass,E. crus-Balli (L.) Beauv.l. Other acylamidases,such as a -inducible produced by Bacillus sphaericus' have broad substratespecificities including action on acylanilide' phenylcarbamate,and substituted phenylurea herbicides. Many rnicrobial hydrolytic enzymesare exfiacellular, thus hydrolysis can occur without uptake. Advances in molecular biology have led to an increased understanding of hydrolytic enzyme actiYe sites, especially those conferring selective specificity' This knowledge will create opportunities for engineering novel resistance mechanismsin plants and biosyntheticand/or degradativeenzymes in microorsanisms.

O 2001American ChemicalSociety 59 Hydrolytic enzymes catalyze the creavage of certain chemical bonds of a by the addition of the componentsof water (H or OH) to each of the frooucts. Many herbicides, fungicides and insecticidescontain moieties t".g., ;ij" or carbamate bonds, or ".rrT carbonyl, yilh phosphoryl and thionyl finU"geO tfrat are subject to enzymatic hydrolysis. A wide array of (amidies, esrerases,Iipases, , peptidases, phosphatases, etc.), with broad and narrow subshate specificities are present in animals, microorganisms and plants. plants and microorganisms should not n€cessarily be expected to possessthe enzymatrc capacity to metabolize xenobiotic compounds. But indeed, ii is ttre muttiplicity of certain enzymes and their broad substrate specificities that make pesti;ide degradarion possible. Thus potential the for hydrolytic cleavageof a given pesticide exists in numerousorganlsms. Various levels of cellular companmentalizarionfor these hydrolytic enzymes exist. Some are cytosolic, while others are associatedwith membrines, mrcrosomes, and other o-rganelles. Some fungi and bacteria also excrete hydrolytic enzymesthat act extracellularly on substrates,and thus pesticide detoxification and degradation may occur without microbial uptake of the compound, Certain hydrolases are constitutive, while others are inducible. The abiliiy of an organism to hydrolyze certain pesticides can render the organism resistantio that com;ouod. Many plants are insensitive to various classes pesticides of due to their unique'mechanism hydrolytic capabilities. Furthermore, differential metabolism is an important in determining selective _tlle toxicity of a given compound among plants and other organisms. Since molecular oxygen is not involvea in hydrolyticlctivity, hydrolysis can occw under anaerobicand/or aerobicconditions. In this chapter we examine the hydrolytic transformations of a variety of pesticide_ chemical classesby plants and microorganisms- The role of theseenzymes in pesticide activation, detoxification and degradition is discussed,and opponunities for exploiting novel hydrolytic rransformation;ofxenobiotics are examined.

Ester Hydrolysis in Plants

Esters are susceptible to hydrolysis by esterases, and to some extent by lipases and proteases. Esterases are ubiquitous in living organisms, and occur as multiple isozymes with yarying substrate specificities and- catalytic rates, For example, fourteen esterases have been isolated from bean (phaseoius vulgaris L.) and seven from,pea (Pisum sativun L.) (,f). The biochemistry and rcle o'f plant esterasesin xenobiotic metabolism has recently been reviewed 1Z;. fl" phyiiological role of thes€ esterases may be multifaceted, e.g., they are involved in fruit ripening, abscission, cell expansion, reproduction piocerres, as well as hydrolysis of ester_ containing xenobiotic molecules. Varying degreesof specificity and kinetic rates are observed among plant esterases. For example, un """tyl "",erur" from mung bean (Vigw.radian) hypocotyls hydrolyzed high molecular weight pectin esters(primary physiological substrates),but could more rapidly hydrolyzJlow molecular substrates such as triactin, and p-nitrophenyl aceta(e(3). 60

Many pesticidesare applied as carboxylic acid esters(Figure l). Since the acid form is generally the active agent, esterasescan play a role in pesticide activation, detoxification, and selectivity in plants (Table I.). Many herbicides have been specifically developedas estersto improve absorptioninto plant tissue,and to alter

Table L Role of Plant Esterases in Herbicidal Activitv in Plants

Mechanism Herbicid.e Plant species Citation Activation Fenoxaprop-ethyl Wheat, Barley, (4) Crabgrass Diclofop-methyl Wheat,Oat, Wild (5) Oat Detoxification Thifensulfuron- Soybeans (6) methyl Chlorimuron-ethyl Soybeans o Increasedabsorption Quincloracesters Spurge (8) andtranslocation Increasedabsorption 2,4-D-Butoxyethyl Bean (e) ester

phytotoxic selectivity. Aliphatic derivatives of the broadleaf herbicide 2,4-D l(2,4- dichlorophenoxy)aceticacidl are widely used.The polar forms of 2,4-D are readily taken up by roots, while long-chain non-polar ester forms (butoxyethyl, and isooctyl este6) are more readily absorbedby foliage. The ability to hydrolyze these 2,4-D estersis widely distributed in sensitiveplants such as cucumber(Cucumis sativus L.) (/0) and tolerant plants such as barley (Hordeum vulgare L.) (,1,1). In a fungicide developmentprogram, ester derivativesof the herbicide4,6-dinitro-o-cresol (DNOC) were assessedas fungicides,and for potential phytotoxicity on broadbean(Vicia faba) (,12). Short-chainaliphatic esters(acetate, propionate and isobutyrates)were readily hydrolyzed and were highly phytotoxic. Howev€r, aromaticesters of DNOC (chloro- and nibobenzoates)were hydrolyzed slowly and exhibited low phytotoxicity. The uptake of (3,6-dichloro-2-pyridinecarboxylicacid) ftee acid was greater compared to that of the l-decyl and 2-ethylhexyl esters in Canada thistle (Cirsizz amense) alndwild buckwheat (Polygonum convolvulus\, and in isolated cuticles of Euonymusfortunei (13, 14 )- De-estedficationwas essentialfor the ldecyl and 2- ethylhexyl es0ersof this herbicideto enterthe phloem and translocateto site of action. The polycyclic alkanoic acid herbicides(PCAs), usually contain more than one ring structue (one is usually a phenyl ring) attachedto an asymmetric,non-carbonyl carbon of an alkanoic acid (,15). Common herbicidesin this class include: diclofop- methyl [methyl ester of (a)-2-[4-(2,4-dichlorophenoxy)phenoxylpropanoicacid], fenoxaprop-ethylfethyl ester of (l)-2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxyl- propanoic acid), and -butyl { (R)-2-[4-[[s-(trifluoromethyl)-2-pyridinyl]- oxylphenoxylpropanoicacid). In plants,PcA-ester hydrolysis, yielding the parent 61

H.OH I O{II2-CO4-CH24H2-O-CH2-CH2€H2-CH3

I

2,4-Dbutoryethyl ester

cl / H-oH V .' -\__/- "-V- o-cH-co-o-cH3 cHs Diclofop-methyl

H-OH 62 acid, is the first enzymatic action on these compounds (15). For the PCAS, de- esterification is a bioactivation, not a detoxification mechanismas demonstratedfor diclofop-methyl (5). The carboxyesteraseresponsible for de-esterificationof the PCA herbicide chlorfenprop-methyt [methyl 2-chloro-3{4-chlorophenyl)propionate] has been partially purified fiom oats (Avezo sativaL.) (16) and wild oats(Avena fatua L.) (,lZ). In tolerant species,the PCA-free acid is detoxified by different mechanisms, i.e., diclofop via arylhydroxylation and subsequentphenolic conjugation (,18), and fenoxaprop, via glutathione conjugation (.19). Rice is generally tolerant to fenoxaprop-ethyl,but under low light intensity, rice can be damagedby fenoxaprop- ethyl treatment (20). However, similar rates of in vitro a.l,din vivo fenoxaprop-ethyl de-esGrificationwere found in rice seedlingsgrown in either, normal light or low light conditions (2,1). Esteraseactivity was also measuredusing fluorescein diacetate (FDA) as a substrate(21). In fenoxaprop-ethyl-treatedrice, FDA esteraseactivity was 4l Vo lower in shaded plants compared to unshaded plants. However, in untreated rice, FDA esteraseactivity was 22Volower in shadedversus unshaded plants. Hence, the phytotoxic compoundsfenoxaprop-ethyl and fenoxapropacid, persistedlonger in shaded plants, which may explain this phytoaoxicty to plants under low light conditions. The herbicide chloramben (3-amino-2,5-dichlorobenzoicacid) was rapidly metabolizedto an N-glucosidein resistantplant species,while sensitiveplans formed the carboxy-glucoseester (22). The glucoseester is unstablein vivo due to hydrolysis by esterases,thus an equilibrium betweenthe esterand free acid is maintained. The selectivity of the sulfonylureaherbicides is basedon several detoxification pathways,including oxidative and hydrolytic mechanisms(6). Soybean(Glycine max Merr.), can de-esteriS thifensulfuron-methyl { methyl 3-[[[[(4-methoxy-6-methyl- 1,3,5-triazin-2-yl)aminolcarbonyllaminolsulfonyll-2{hiophenecarboxylate} to the fiee acid (non-phytotoxic), but soybean is unabl€ to de-esterify the herbicide analog, metsulfuron-methyl { methyl 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-amino]- carbon-yllaminolsulfonyubenzoatelwhich injures the crop plant. Soybeanesterases can hydrolyze thiophene O-carboxymethyl esters and O-phenyl€thyl este6, but not O- phenyl methyl estersof certain sulfonylureas(6). Chlorimuron-ethyl { ethyl 2-[[[[(4- chloro-6-methoxy-2-pyrimidinyl)aminolcarbonyllaminolsulfonyll-benzoatel detoxif- icaction occuns via de-esterification of the ethyl group and dechlorination via homoglutathioneconjugation in soybean(Z). However, conjugationoccurs three-fold more rapidly than de-esterification. A recent study compared the phytotoxicity of thirteen ester dedvatives (C5 to Cl6) of the herbicide (3,7-dichloro-8-quinolinecarboxylic acid) for phytotoxicity against leafy spurge (Euphorbia esuls L.) (8). Foliar application of quinclorac caused rapid death, whereas quinclorac esters applied at higher concentxationsto foliage causedonly phytotoxicity, not mortality. When applied to soil, quinclorac esterswere metabolizedby a seriesof and oxidations while hydrolysis was limited. This resultedin the slow releaseof quinclorac which increasedherbicide efficacy against leafy spurge plans. 63 Ester Hydrolysis in Microorganisms

Studies of fenoxaprop-ethyl (23, 24) and diclofop-methyl (25) degradation in soils demonstrated a rapid hydrolysis of both compounds to their parent acids. This hydrolysis was more rapid in moist and non-sterilesoils compared to dry or sterile soils, which suggestedmicrobial degradation. The role of enzymatic hydrolysis of diclofop-methyl was reduced with certain microbial inhibitors (propylene oxide and sodium azide) (25). During hydrolysis of diclofop-methyl and fenoxaprop-ethyl in soil, enantiomeric inversion was observed (2O. The S enantiomers of both compounds underwent a more rapid inversion than the R enantiomers,and rates of inversion were dependenfon soil type. No inversion was observedwith entantiomers of the parent compounds. Fenoxaprop acid undergoes funher degradation in soil, forming metabolites such as Gchlorobenoxazolone,4-[(6-chloro-2-benzoxazolyl)- oxylphenetole, and 4-[(6-chloro-2-benzoxazolyl)oxy]pbenol(24). Soil pH also affecs the degradation pathway of fenoxaprop-ethyl. Under acidic conditions, the rate of de-esterification was signifrcantly lower than under neutral soil conditions, however, the benzoxazolyl-oxy-phenoxy ether linkage of fenoxaprop-ethyl was prone to non-enzymatic cleavage under acidic conditions (22). De-esterification of fenoxaprop-ethyloccurs readily in mixed microbial cultures (28) and in pure cultures and enzyme extracts of bacteria, especially fluorescent pseudomonads(27, 29). Fenoxaprop-ethylde-esterification in bacterial enzyme preparationsis pH sensitive, with the highest activity in the neutral to slightly alkaline range. The same Pseudomonas strains that hydrolyzed fenoxaprop-ethyl were unable to de-esterify chlorimuron-ethyl (R.M. Zablotowicz, unpublished results). Four distinct types of esterases are found in a P. fluorescens strain (30). These P. fluorescenJ esterases differ in substrate specificity, cellular location and structure. Esterases have been cloned, and proteins have been sequenced from several microorganisms,e.9., two fgrulic acid eslerasesfrom Aspergillus tubingensis(3l), a cephalosporin esterase from the yeast Rhodosporid.ium toruloides (32\, a chrysanthemic acid esterase frcm Anhrobacter globiformis (33), and several other esterases from Pseud.omonasfluorescezr strains (30, 34, 35). The ability of these €sterasesto hydrolyze ester linkages of pesticides has not been examined. Since molecular oxygen is not involved, enzymatic hydrolysis can occur under anaerobic and aerobic conditions. The of prokaryotic and eukaryotic est€ras€scontains the sedne motif (Gly-X-Ser-X-Gly), originally characterizedin serinehydrolase (3O. This conserved peptid€ is part of a secondarystructure of the enzyme molecule located between a - strand and an a-helix (32). A general mechanism has been proposed for the catalyric activity of the esterasesuperfamily (Figure 2). This mechanisminvolves the formation two tetrahedral intermediates(.t/), with the active serine serving as a nucleophile enabling ester bond cleavage. A histidine residue in a p-srand, is also involved in formation of an ionic attachment at the carbonyl oxygen atom of the substrate during catalysis. Reaction of the second intermediate, with a molecule of water, concomitantly releasesthe parent acid of the subsrate and regenemtesthe active serine of the enzyme. A similar reaction mechanism is postulated for the serine 64

proteases. However, a chrysanthemicacid esterasefrom Az& robacter sp. is similar to many bacterial amidasesthat possessthe Ser-X-X-Lys motif in the active site. This esterasemay provide a unique opportunitiy for biotechnological synthesis, since it stereoselectivelyproduces (+)-r-chrysanthemicacid, used in pyrethroid insecticide synthesis. Certain esterases,e.g., the R. torulaides cephalosporin esterase, also have acetylating actiyity when suitable acetyl donors are present(-t2). The R. toruloides cephalosporin esterase is a glycoprotein (90 kDa glycosylated; 60_66 kDa de_ glycosylated)with eight potential binding sites(Asn-X-Ser or Asn_X_Thr)for glucose. When de-glycosylated, enzyme acdvity was reduced about 507o, indicaiing an important role for glycosylation in stabilizing the natiyeprotein structure. Feng et al. (38) transformed tobacco (Nicotiano tabacum L.) and tomato (Lycopersiconesculentum L-) with genesencoding for rabbit liver esterase3 (RLE3). This esterasegene expressedin th€se plants, confened resistanceto the herbicide thiazopyr Imethyl 2-(difluoromethyt)-5-(4,5-dihydro-2+hiazolyl)_4_(2-methylpropyl)- 6-(trifluoromethyl)-3-pyridinecarboxylatel.These researchers proposed, that a critical assessmentof microbial esterasesmay provide other hydrolytic enzymes that are useful in conferring pesticide (herbicide) resistancein plants. Such enzymes could also play a role in reducing the levels of pesticides,their metabolites, and other potentially harmful xenobioticsin food products.

Inhibition of Esterasesin Plants and Microorganisms

The effects of two fungicides, capran lN-(trichloromethylthio)_4_cyclohexene_I, | _ dicarboximidel and folpet [N-(trichloromethylrhio)phrhalimide],and a suftrydryl binding inhibitor, perchloromethylmercaptan,were assessedon esteraseactivity of Penicillium duponti (J9). Esteraseactivity usingp-nitrophenylpropionate as subsrate was inhibited 50Vot:y all three compoundsat 0.5 to 2.0 uM. But, concentrationsthat totally inhibited p-nitrophenylpropionateesterase activity had no effect on c_naphthyl acetateesterase activity. The extreme sensitivity of certain fungal esterasesto these two fungicides, suggesrsthar part of their toxicity to fungi may be due to esterase inhibition (39).

Amide Hydrotysis in plants

Amide and substituiedamide bonds are presentin severalclasses of Desticides,i.e.. acylanilides; [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide]; carboxin (5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide); diphenamid 2-chloro-y'r'-t -merhyl.2-merhoxy)elhytl-r'V-d [ [( (2,4 imethyt-Lhien-3-yl] ; metaiaxyl l/V- (2,6-dimethylphenyl)-N-(methoxyacetyl)- merhylesterl; [2-chloro- N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamidel; and propanil, [N-(3,4-dichlorophenyl)propionamide],phenylureas: diuron [N-(3,4-dichlorophenyl)_ I 65

NJv-dimethylureal; fluometuron {N,N-dimethyl-N-l3-(trifluoromethyl)phenyl]urea]; and linuron, [N-(3,4-dichlorophenyt)-N-methoxy-N-methylurea];and carbamates:IPC (isopropyl carbanilate); and CIPC (isopropyl rz-chlorocarbanilate). Some structural examplesofthese substitutedamides are given for comparison(Figure 3.) Rice (Oryza sativa L.) is tolerant to the acylanilide herbicide propanil, due to the presenceof high levets of aryt acylamidase(EC 3.5.1.a), which hydrolyzes the 'fhis amide bond to form 3,4-dichloroaniline (DCA) and propionic acid (40' 4l). enzyme activity is the biochemical basis of propanil selectivity in the control of barnyardgrasslEchinochloa crus-galli (L.) Beauv.l in rice. Barnyardgrasstissue was unable to detoxify absorbedpropanil due to very low enzymatic activity; rice leaves contained sixty-fold higher aryl acylamidaseactivity than barnyardgrassleaves (4O). Propanil aryl acylamidaseactivity is also widely distribuied in other crop plants and weeds(42,43\. Plant aryl acylamidaseshave been isolated,and partially purified and characterized ftom tulip (Tulipa gesnariana v.c. Darwin) (44), dandelion (Taraxacum officinale Weber) (45), and the weed red rice (OryU sativa L.) (4O. Red rice is a seriousconspecific weed pest in cultivated rice fields in the southernU.S. (42)' and its ability to hydrolyze propanil limits the utility of this herbicide where red rice is present. Propanil hydrolysis by aryt acylamidases has also been observed in certain wild rice (Oryza) species(48). With intensiveuse of propanil in Arkansasrice production over a thirty-five year period, bamyardgrass(initially controlled by propanil), has evolved resistanceto this herbicide, and this biotype is currently a serious problem (49). Populations'of propanil-resistantbarnyardgrass have been verified in all southernU.S. statesthat use propanil in rice cultivation. A series of experiments with propanil-resistant barnyardgrass showed that the mechanism of resistance was increased propanil meiabolism by aryl acytamidaseactivity (50,5/). Increasedpropanil metabolism by aryt acylamidase was also shown to be the resistance mechanism in another related weed,junglerice [Echinochlaa colona (L.) Link] (52). Naproanilide [2-(2-naphthytoxy)-propionanilide], a herbicidal analog of propanil, was hydrolyzed by rice aryl acylamidase (53). The cleavage , napthoxypropionic acid is phytotoxic, however, it is hydroxylated and subsequently glucosylated as a detoxification mechanism in rice' Naproanilide hydrolysis also occurred in a susceptible plar\t (Sagittaria pygmaea Miq.), but napthoxypropionic acid was not metabolizedfuther in this species(53). Initial assessment of substrate chemical sEuctwe and aryl acylamidase activity hasbeen studied in enzymepreparations from variousptants, e.g., rice (40)' tulip (44)' dandelion (45), and red rice (46). Nine mono- and dichloro-analogsof propanil were examined for substrate specificity of these enzyme preparations. Different profiles of hydrolytic rates were observed among species. In all species tested (not tested in red dce), little or no activity was observed with 2,6-dichloropropionanilide. In rice' greater activity was observed with 2,3-dichloropropionanilidecompared to propanil; *hil" in tutip, equal activity was observedwith propanil, 2,4-dichloropropionanilide' and 4-chloropropionanilide as subskates. In a similar fashion, the effects of alkyl chain length on 3,4-dichloroanilide subsaateswere evaluated. Propanil was the best substrate for all four enzyme prepantions. Reducing the chain length to one carbon 66 o o- ll I Tetrahedral R-O-C-R* ------R-O-C-R. Intermediate I + r-ort Ewvu,Ser/ ",t,^*JO\ o tl O_C_R++ R_OH /.- f,,ra,ranSer/ lllrO o 7 ll OH R.-C-O- <-- I + -oH /-o-!-*- #ffi#:" Ev,rwser ,/ E"raa.rSer/ 6- Figure 2- Scherutticmechanism of esterase-mediatedhydrolysis-

HO Il N-C-OCH(CHt2 /N" ll lPropham \/ HO |ll T-N(cHr, H.OII Linuron

OH OH ctl3o{-N\f\./,,ill tT>ay.",ill ll I Ho-H ll I \/ \/ Phenmedipham

Figure 3. Selected pestici.deswith qmide and substituted amide bonds. 67

propanil' (i.e., the acetamideanalog), decreasedactivity by 18 to 50%, compared to activity by 60 i""."^i"g the atkyl chain-lengthor inserting alkyl branching' reduced to l@9o. Aryl acylamidases from several plant species have been purified to homogeneity (54). The enzymes from orchardgrass and rice are quite similar' Both have molecular bound' These enzymes share a comnon *eights"op,i-utn of about 150 kDa, and are membrane for propanit' and are both inhibited bv the fff of 7.0, similar Km's from Monterey ins""ti"ia" carbaryl. A gene for aryl acylamidase has been cloned of 319 amino pine (Pinzs radiata) male pine cones (55). This protein, comprised site' acids, is similar to esterasescontaining a serinehydrolase motif in the active

Amide Hydrolysis in Microorganisms

in diverse As we have reviewed (56), aryl acylamidaseshave been characterized pesticide speciesot atgae,bacteria and fungi, and propanil has beenthe moststudied enzymes .uU"au,". itt" distribution of aryl acylamidasesand related hydrolytic in produced by selected microorganismsare summarized(fable II)' Differences generaand Iubstrate specificity and inducibility of theseenzymes are found-among speciesof'microbes. For example,aryl acylamidasesproduced.by.P' fluorescens (propanil' sirainsRA2 and RB4 had a substrate.ung".ii^it"a to certainacylanilides herbicides nitroacetanilide,and acetanilide),and were ineffective on several (linr'non diuron)' a contuining substituted amide bonds: phenylureas --and pft"nyf"u.iurnt (CIPC), chloracetamides[alachlor and the N{ealkylated metabolite !-chioro-ll-(2',6-diethylacetanitidel, and a benzamide{pronamide -[3'5-dichloro(N- are found i,r -Jirr.,fryi-i-p-pynyl)benzamidel) (60. Similarsubstrate -specificities have activity in oitr". U*t".iut slains (fable IU)- Other microbial acylamidasesalso restrict€dbo certain acylanilidis, e.g.,Fz sarium orysporum(69)' Bacillus Some acylamidaies,such as a linuron-inducibleenzyme produced by (60)' have wide ,pno"riir, (5i), and an extracellularcoryneform aryl acylamidase phenylcarbamate' r'uUrtut" tp""lfr"ities includinghydrolytic action on acylanilide' are two and substitutedphenylurea pesticides. ln a Fusariumorysparum strain' there induced by p- distinct aryl acylamidases:one induced by propanil, the second metabolism of "t foroptr"nyf methyl carbamate(69). One study has compared-the were ""u"rui ptt"nytu."u herbicidesby bacteriaand fungi (70)' Nine fungal species l- ,no." "if""tiu" in degradinglinuron or isoproturon[3-(4-isoPropylphenyl)-l ' N or O- Jimethylureal"o*purJd to fenuron (l,l-dimethyl-N-phenylurea)'-but Only one of five a""itvl'",." i"., ttyirolysis) wasthe major degradationmechanism'- of 3'4- bacterial isolates (a psiudomonad)*"tuUotit"O linuron with the formation The hydrolytic dichloroaniline,indicating hydrolytic cleavage of the urea bond - has also been ,n""t unir. for phenylurJaherbicides by a ioryneform-likebacteria showr with un organis- capubleof hydrolyzinglinuron > diuton>- [N- li-"rrroroptt"nyr;i-methoxy-N-methylureal >> metoxuon [N-(3-chloro-4-methoxy- phenyl)-N,N-dimethylureal>>> isoproturon(7'l)' 68

Table II. Selected Microbial Speci€s that Produce Aryl Acylamidases and Related HYdrolYtic EnzYmes

Substrate Inducer Citation Anacystis nidulans CIPC,IPC nt* 6n Aspergillus nidulans Propanil, Constitutive (58) . (propionanilide**) Bacillus sphaericus Linuron, carboxin,IPC Linuron (5e) CoryneformJike, CIPC, linuron, (Acetanilide) (60') strain A-l naproanilide, propanil Fusariun oxysporum Propanil,CIPC, Propanil and (6t) (acetaniI ide) phenylureas Fusarium solani Propanil,(acetanilide) Propanil and (62) (acetanilide) Nostoc entophytum Propanil nl (63) Penicillium sp. Karsil, propanil, Karsil (64) (acetanilide) Pseudomonas (Acetanilide), (Acetanilide) (65) fluorescens (p-Nitloacetanilide, p-Hydroxyacetanilide) (66) P. Jluorescens Propanil,(acetanilide, Constitutive nirroac€tanilide), P. picketti Propanil Constitutive (6D P. striata cIPc, IPC, cIPc (68)

NOTE:*nt = not tested;** compoundsin par€nthesisare not usedas pesticides'

Propanil hydrolysis yielding DCA (72,7J) is the major mechanism for dissipation of this compound in soil. Many miooorganisms hydrolyze propanil to DCA, and enzymes from several species have been isolated, partially purified' and characterized. For example, ninety seven bacterial isolates were collected from soil and flood waier of a Mississippi Delta rice field over a two year period following propanil application (Table IV). Overall, 3770of the soil and water isolatesexhibited propanit hyirolytic activity. Although activity was observedin both gram-positiveand gram-negative isolates, there was a greater frequency of propanil-hydrolysis among of several of these lram-negative bacteria. The hydrolytic activity of cell-free extracts isolates, and other rhizobacterial cultues on several substrates,was assessedusing metbods described elsewhere (66). Only acylanilides were hydrolyzed, with no detectable hydrolysis of carbamate and substituted urea herbicides (Table V)' All isolates, excipt AMMD and UA5-40, were isolated from soil or water that had been previously 69

-6_cE 9eI EEEEEEEEEE at) q A'c E* A ;a:€eE5 9=vY=Y==r- E 6<: F E] tJ

9q", h 9p: 3 3 - E S'E E :;FE ER;*s5:i:a.>.E€ F(o !\ E >,4 \J{=!-

aOJ z =-k: *s:=n i EEiE 6r5: e;! i (a i F;P -^-9o3-,r,o.+ **=3:*;:!: 9a*a eg<& -!tr ,< .Fo<9 q>&e&&&/

ii.; .= ; ii-$ ,: ^ O:i E:? €;;"-.: t$tJ*Dss$d..11E- I 5 >2 tr ^ P gF,6 9-Pts5' pEsF![ '6 i.: I a't -33U33F 4::l-:; F iesSFFn xs600C)0 .[iEr ii's s I s i\ .ii -E o E e *5-:-\ir-:: tr 'h F ,sil * {a.idq.dQ ogdx 266i 70

Delta Table IV. Recoveryof Propanil-Hydrotyzing Isola&s from a Mississippi Rice Soil and Flood Wattr

Source Gram'stain Propanil- Tonl Isolates reactLon isolates tested 1982 Negative I IJ Positive 2 t9 Water Negative 4 8 Positive o l5 o 1983 Soit Negative Positive 3 ll Water Negative 3 8 Positive 4 l4 Total soil l9 52 Total, water t'l 59 Total $am-Positive 15 Total gram-negative 2l 38

following NOTE: Bacteriawere isolatedon tryptic soy agu (lo%)' from soil and warcr tested propanilapplication. Individualcolonies werJ subcuhured, ascertained fo! purity and (log ml; 50 io. b.". ,iuin ,"""tion. Propanilhydrolysis assessed in cell susp€nsions I l 0 cells and -M pot^"iu. phosphate,ptl S-o;S0O llvl propanil)after 24 h incubation' Propanil metatolites were determinedby HPLC as describedelsewhete (66)'

aryl exposed to propanil. P. Jluorescens strains RA-2 and RB4 exhibited organlsm acylamidase activity several orders of magnitude higher than any other isolated in our studies(56,66). The ability to hydrolyze propanil was studied in fifty-four isolates of fluorescent pseudomonadsiollectedf;omihree MississiPpiDelta lakes (74)'- Overall' about 607o bacteria were of the irolate" hydrolyzed propanil, and all the propanil-hydrolyzing P' identified u" P. luor"tr"ni biotype II. Most propanil-hydrolyzitg fluorescens isolatestransformedDcAto3,4.dichloroacetanilide'Thepotentialforacetf not be activity by these P, fluorescens aryl acylamidases should (75\ and overlooked, since the enzyme iiolated from Nocardia globerula amidase from Pseudononas lcidovarans (76) possesses this activity The This Rhodococcussp. strain R3l2 has both amidaseand acyl transferaseactivity on acyl enzyme was expressed in Escherichia coli and utrlize'd in kinetic studies fiom amides transferase aci;ity gn. This purified enzyme catalyzed acyl transfer acetyl and hydroxamic acids to onty water or hydroxylamine' Funher aspects of and tranrfe.""" activity will be addressedin the later presentationon bialaphos phosphinothricin' detoxifi cationhesistance. i variety of methods (radiological, sPectophotometdc, and HPLC) are available measuring aryl to ,n"*ur" "n"yrnutic hydrolysis oi amides. A colorimetric method for *T:":ltll acylamidaseiivity in soil using2-nitroacetanilide (2-NAA) *,t:qt:.ut 7l

raC-Propanil Table V. Metabolism by Cell Suspensions of Soil, Water and RhizosPhere Bacteria

Genera C Recovered in methanolic extracts DCA 3,4-DCAA '11.1 Bacillussp. S9282 S nd 10.5 18.5 Flavobacterium w92B14 w nd 88.6 4.5 6.9 sp. P. cepacia AMMD R 74.5 6.9 18.6 Nd Nd Nd P. fluorescens RA-2 R nd 100 5.0 t.5 P.fluorescens RB.3 R 2.5 91.0 Nd Nd P.fluorescens RB-4 R nd r00 Nd Nd P.fluorescens UA540 R 100 nd '70.3 4.2 16.3 P.fluorescens w92812 w 9.2 Rhodococcus S92A1 S 15.9 to.2 3.5 Nd sp. Rhodococcus s93A2 nd 96.r 3.9 Nd

= highly NOTE: DCA = 3,4-dichloroaniline;3,4-DCAA = 3'4-dichloroac€tanilide;Origin isolate:R polarmetabolites limmobile in benzene:acetone solvent): S = soil isolate;w= water = rhizosphereisolate; nd = nonedetected. cell suspensions[48 h tryptic soy broth cultures,log propanil(800 r iO ""rL rnr't; potassiumphosphate buffer (50 mM, pH 8'0)l weretreated with Propaniland metabolites identified trM, s.0 kBq mi-') andiniuuaied at 28"C,l5orpm,24h by TLC and radiological scanningas described elsewhere (56)'

(5 hydrolytic enzymes such as alkaline phosphaase (3 to 5%)' and aryl sulfatase to t\vo'..' tryt acylamidaseaciivity was 1.3- to 2.5-fold higher in surface no-till soils greater comoared io conventional-tilled soils. This would be expected due to the microbial populations and diversity associated with the accumulation of soil organic matter in tirelu.face of no-till soils. Maximal aryl acylamidaseactivity was observed at pH ?.0 to 8.0, and activity was reduced at assay temperatures above 3loC Therrnal inactivation has also been reported for a purified aryl acylamidase from P' Jluorescens (65). With respect.tochloroacetamide herbicides' there is only limited evidencein the literature for cleavage of the substituted amide bond The fungus Chaetonium (79) globosum was show-n to hydrolyze substituted amide bonds of alachlor and -methylethyl)-N- iretolachlor (80). A unique cleavageof [2-chloro-N-(l phenylacetamidel at the benzyl C-N bond, by a Morarclla isolate' has been i"rnon.ttut"d (8i). However, little is known about this mechanism,or iLsdist bution among other species. Recently, two bacteria (Pseudomonas and Acientobacter) initially capabl of metabolizing ptopuihlot were described (82) Both strains "fhe then de'halogenated propachlor io N-isopropylacetanilide. Acientobacter strairj' ring hydrolized the'amide bond, befori thi releaseof isopropylamine,and prior to via N- "i"urug". T1r- Pseudomonas sfiain initially transformed propachlor '1, dealkylation. This metabolite was then cleaved at the b€nzyl C-N bond as in the Moraxelle isolate described above (8/). Aryl acylamidaseshave been purified from several bacterial species including, B- sphaericus (59), a coryneformlike bacterium (60), Nocqrdia globeruln (75), P. aeuriginosa (83), P. fluorescens (65), and P. pickeuii (62). These various enzymes have been shown to be quite diverse. Among thesefour genera,the aryl acylamidases ranged in size from 52.5 kJ)^ for the P. Jluorescens enzyme, to about 127 kDa for the coryneform and, N. globerula aryl acylamidases. The P. pickettii enzyme is a homodimer, while the other euymes are monomers. A novel amidasefrom P. putida, specific for hydrolyzing N-acetyl arylalkylamines,was purified to homogeneity (84). This protein (MW =150 kDa) is a tetramerof four identical subunits. This enzyme hydrolyzed various rV-acetylarylalkylamines containing a benzeneor indole ring, and acetic acid arylalkyl esters,but not acetanilidederivatives. To our knowledge, no genes for specific pesticide-hydrolyzingalyl acylamidaseshave been cloned. A multiple alignment and cluster analysishas been performed on amino acid sequences of 2l amidasesor (85). A hydrophobicconserved motif [Cly-Gly- Ser-Ser (amidasesignature)l has been identified which may be impofiant in binding and catalysis. Amidases from prokaryotic organisms also have a conserved C- terminal end, not found in eukaryotes. These studies also indicate similarities of amino acid sequencesamong amidases,nitrilases and . Metals, i.e., Hg*, Cu* Cd*and Ag*, that affect sulftrydryl groups differentially inhibited bacterial aryl acylamidases(59,60,65,83). EDTA did not inhibit the 8. sphaericus (59) and P- fluorescens (65) enzyraes, but 2.5 mM EDTA inhibited the coryneform-like aryl acylamidaseby about 90% (60). An aryl acylamidasefrom P. aeuriginosa required Mn* or Mg* (83), but thesecations were not required by other enzymesmentioned aboYe. Initiaf studiesofrhe interactionof chemical structureand microbial (Penicillium sp.) aryl acylamidaseactivity were evaluatedin an enzymeinducible by karsil [N-(3,4- dichlorophenyl)-2-methylpentanamidel(64). Activity was greater with longer alkyl amide substitution, i.e., activity was 4, 262 and 1000 units for acetanilide, propionanilide, and butryanilide, respectively. Of the six herbicides evaluated, propanil had the highest activity (520 unis), comparedto karsil (70 units), solan [N- (3-chloro-4-methylphenyl)-2-methylpentanamidel (55 units), dicryl [N-(3,4- dichlorophenyl)-2-methyl-2-propenamidel(40 units), and no activity was detected with diuron and CIPC. The hydrolysisof sevenpara-substituted acetanilides by three bacterial species (Arthrobacter sp., Bacillus sp., and Pseudomonas sp.) was compared to rate constants for alkaline-mediatedhydrolysis (86). In these studies, para substitution did not affect acetanilide hydrolysis by resting cells of these bacteria, however alkaline-mediatedhydrolysis was highly affe.ted. These studies suggest different intermediatesand mechanismsfor biotic versusabiotic transformations. A structure-activity study evaluated acylanilide herbicide chemical structure using model substrates and four bacterial strains [two Arthrobacter spp. (BCL and MAB2), a Corynebacteriumsp. (DAK12), and an Acinetobacter sp. (DVl)l capable of growth on acetanilide(82). When the nitrogenof acetanilidewas alkylated (methyl or ethyl), the compound was an unsuitable subsnate for all four strains. When a para- 73

methyl group was presenton the acetanilidering, activity occurred in all strains,but was lower than acetanilide activity in DVI and one of the Arthrcbacter sl'Jairls, (MAB2). When the methyl group was in the onho or meta position,activity similar to that on acetanilide occuned in two strains (DAKI2 and BCL), but was only about 3OEoof rhe acetanilide rate in MAB2, and undetectablein DVl. Little or no activiry in all four shains was found with dimethyl substitution in the 2 and 6 positions. Consequently,alachlor and metolachlor(with 2 and 6 alkyl substitutentson the ring) are mor€ persistentin soil comparedto propachlor with an unsubstitutedaniline ring (88). The fungicide ipridione [3-(3,5-dichlorophenyl)-l{-isopropyl-2,4-dioxoimidazo- lidine- I -carboxamidel undergoes several potential amide hydrolytic .eactions. Ipridione degradationby an Anhrobacterlike strain (89) and three pseudomonads(P. fluorescens, P. paucimobilis, and Pseudomonassp.) has been reported (90). Initial cleavage of ipridione forms lr'-(3,5-dichlorophenyl)-2,4-dioximidazoline and isopropylamine- The imidazolidine ring is cleaved, forming (3,5-dichloro- phenylurea)aceticacid, which can be further hydrolyzed to 3,5-dichloroaniline. 3,5- Dichloroaniline is a major metaboliteobserved in soils wheremicroflora have adapted the ability for enhancedipridione degradation(91).

Inhibition of Plant and Microbial Amidases

Propanil hydrolysis is inhibited by various carbamate and organophosphale insecticidesin plants (40, 92) and microorganisms(93). Competitive inhibition of aryl acylamidaseactivity by thesecompounds was the basisfor increased(synergistic) injury to rice, caused when insecticideswere applied to rice in close proximity to propanil application (94). Synergisticeffects of propanil with severalagrochemicals: carbaryl (1-naphthyl N-methylcarbamate);anilofos IS-[2-[(4-chlorophenyl)(1-methyl- ethyl)-aminol-2-oxoethyll O,O-dimethyl-phosphoro-dithioate); [N-( l - €thylpropyl)-3,-dimethyl-2,6-dinitobenzenaminel;and piperophos {S-[2-(2-methyl-l - piperidinyl)-2-oxoethyuO,O-dipropyl phosphorodithioate)in propanil-resistantbarn- yardgrass were recently detected using a chlorophyll fluorescencetechnique (95). Carbaryl's synergismis due to its compeiitive inhibition of aryl acylamidase(40), but the exact mechanism of the other synergistsin propanil-resistantbarnyardgrass is presentlyunknown. Interactions of insecticidesand soil aryl acylamidaseactivity have also been reported (93). When soil was treatedwith p-chtorophenylmethyl carbamate,propanil hydrolysis was substantially inhibited, and subsequent formation of tetrachloro- diazobenzenewas reduced l0- to 100-fold. Carbaryl at t00 pM inhibit€d propanil aryl acylamidase activity by 1O to 70% in several bacte al strains (66, 96\. The Fusarium solani propanil-aryl acylamidaseswere insensitiveto high concenrations of carbaryl and parathion (O,Odiethyl-O-4-nitrophenyl phosphorothioare),however the chloroacetanilide,herbicide ramrod (N-isopropyl-2-chloroacetanilide),competitively inhibited acetanilidehydrolysis. 74

CarbamateHydrolysis in Plants

Carbamateshave a broad spectrumof pesticidalactivity and include commonly used insecticides, herbicides, nematicides (aldicarb {2-methyl-2-(methylthio)propanalO_ [(methylamino)cabonyl]oxime] and fungicides. There are three major ciasses of carbamates: methyl carbamates: aldicarb, carbofuran (2,3_dlhydro-2,2-dimethyl-Z_ benzofuranol methylcarbamate),and carbaryl; phenylcarbamates:CIpC, IpC, and phenmedipham; and thiocarbamates: EpTC (S-ethyl dipropyl carbamothioate), I butylate [S-ethyl bis(2-merhylpropyl)carbamothioate],and vernoiate(S-propyl diprop_ I ylcarbamothioate)- The behavior of insecticidal carbamateshas been extensively reviewed,(97, 98). Studies on IpC (99) and CIpC (100, I1t) in planb indicate thai the major metabolic route is aryl hydroxylationand conjugation. plants do not cleaye the carbamate bond of the phenylcarbamab herbicides, which is distinct from the initial metabolism in either microorganismsor animals. The thiocarbamatessuch as EPTC are metabolized in tolerant species such as corn, cotton (Gossypium hirsutum), and soybeansvia initial oxidation to the sulfoxide followed by glutathioneconjugation (102, IO3). The pathway for EPTC metabolismin olants is similar to that found in mouseliver microsomes(,104).

Carbamate Hydrolysis in Microorganisms

Microbial degradation of carbamates occurs readily in soil. Accelerated degradation of certain carbamateinsecticides has led to ineffectivenessof thesecompounds, e.g., control ofphyloxera in vineyardsby carbofuran(.105). Bacterial isolatesfiom several geneta (Art hrobacter, Achromobacter, ATospirillum, Baci(us, and,pseudomonas) can hydrolyze various insectioidalcarbamates (_/06). Hydrolysis is the major pathway for the initial breakdown of carbofuran, but liule is known about the fate of the metabolitesformed by this mechanism(,/06). Mineralization ofthe carbonyl group of carbofuran occum more extensively comparedto mineralizationof the ring struciure (107). The toxicity of aldicarb is greatly reducedwhen it is hydrolyzed to the oxime and nitrile derivatives (108), but oxidation of aldicarb to the sulfone or sulfoxide yields compounds with similar or greater toxicity. Organophosphoruscompounds: paaaoxon(phosphoric acid diethyl-4-nirophenyl ester), chlorfenvinphos [phosphoric acid.2-chloro- l -(2,-4-dichlorophenyl)ethyldiethyl esterl, and disulfoton (phosphoro_ dithioic acid O,O-diethyl S-[2-(etflthio)ethyl]ester] which are esterase inhibitors, suppressedcarbofuran hydrolysis in soil for 3 to 2l days (109\. Tlte persistenceof carbofuran was increased when these esterase inhibitors were combined with the cytochrome P-450 inhibitor, piperonyl butoxide. This synergism is due to rhe inhibition of both major mechanismsof carbofurandegradation, i.e., hydrolysis and oxidation. Some microbial aryl acylamidases fp. striata (l l0), B- sphaericus (59) and a coryneform-like isolate (60)l can hydrolyze certain phenylcarbamates,e.g., CIpC and IPC, as summarized in Table II. However, the p. striata aryl acylamidase is unable to hydrolyze methylcarbanates such as carbaryl. Carbamate hydrolases such as the 75 Anhrobacter phenmedipham-,is specific for phenylcarbamates whereas . (1/,/), the Achromobactercarbofuran-hydroiase i. .p""iFo-iJr-_"tnylcarbamates (1 12\. carbamate_hydrolase .,,^,A jloroli" has beenpurified from a pseudomonassp. (.1,13).This enzym€is composedof two identicaldim"r" *i,f, u ilol""utu. weightof 85 kDa each,and is activeon carbaryl,carbofuran "na "fai"-i a, ,uUstrates(,flj). TheAchromobacter carbofuran-h)droia.. t ^ U""npurinea oioriogen"ity anOt as" molecularweight of 150 kDa. This enzymeis eithercytoplasmic-or occurs in the periplasmicspace, aDd requiresMn* asin actiuatortrlil.'rt rrusno ureaseactivity and doesnot hydrolyze benzamide.The genesro, it. irnroii*rer carbofuran- hydrolasehave been cloned,however they werepoorly expressedrr many gram_ negativebacteria such asE. coli,Alcalig"ni, "utroph^, i"a i. prAa". This indicates *":Iuo processingis llu, requiredtJ produce;fil;;"i;;;. The for phenmedipham-hydrotase(pcd) senes ha-vealio.been cloned unO tf,"'fiot"rn purified to homogeneity_(///). The phenmedipham_hydrolase i, u rno"or"i'ii,r, a molecular weight of 55 kDa, and conrainsrhe esterasemotif -u-iii,ionut phenmedipham-hydrorase fC-f-yii".-X_Cfyl. t" arso has hydroryticactivity o; ,i" -t "r,"ru"" substrate,p-nitrophenylburyrat€. -fhe pcd gene rv". "*p.""r"J-than tobacco, and conferredresisrance to phenmedipham'u, .ui.. I O_fofJfiigi., normat field applicationrares (/1J). Arrhoughbacteria have been irorut'J- tnui porr"ss diverce hydrolytic degradationmechani-sms for cartraryl,few organismsare capableof completemineralization of the entiremolecule. Whena b-acterialconsortium (two Pseudomonasspp.) wasconstructed, both hydrolysis""J-"r"ii"'m**lization of carbaryloccuned (/16). Thiocarbamateshaye been developedas herbicides(butylate, EpfC and vernolate)and fungicides. Repeatedapptiiation of rnesecompouiA's to soit led to the uevcropmentol mtcrottal populationswith acceleraledrhiocarbamab degradation capability. Soils adapbd to EPTC degadati"n a.o *piOfy O"gruded a related thiocarbamate,vernolate (t lT). However, soils adaptedto U"tyfuti aia "ot .ffiy E?IC and yernolate feerafe U tn. EmC metatoiism o""u.i'in-rnuny genera tnc.tena (Anhobacter, of Baci us, Flavobacteium, pr";ir;;;: ^; Rhod,ococcus um,,paeciromyces, penici ) T1.,jl3'_1?-"11, ium) l1/ay- iJ- nyorolyticand oxrqauve mechantsmsh-ave proposed been for EpTC degradationdue to the fact that qrpropropyfamrnewas tound in the media of Arthrob(lcter and Rhodococcus svuns TEI (,//9) and BEI (.f20). Evidencefrom anothetRhodocc-"^.ouin fet, inOi"ua"" ll*,ljrl:tl1?:l.of.the nlon1lgroup of Eprc, foilo;;; ;y-;-dearkylation, rormrng propionaldehyde and N_depropyl_EpTC (121). p-450s, t:.qo"t_'91"- Cytochrome f.o-r Ndealkylation,havi beenctoned from iii.iit nnoao"o""^ strains(122-124\. _!mc

Hydrolysis of OrganophosphateInsecticides by plants

Organophosphateinsecticides { parathion, malathion [S-I ,2-bis(ca6ethoxy)ethyl-O- O-dimethyl dithiophosphatel, and coumaphos (O-3-chloro-4-methyti'-oxo_Ztl_

H 76

chromen-7 yl O.O-diethyl phosphorothioate)l have replacedmany of the chlorinated fnsecrfcrdes,because they are more effective and less persistent. Wheat (Triticum sorghvm (Sorghum :e:tivu:n -L:) :rd vulgare L.) rapidly degradedimethoat€ deimethyl-.S-(/V--methylcarbamoylmethyl)phosphorothiolothion'"t"1 [O,O_ to froOu"ts, *t;"t sugg€stshydrolytic metaborism(,r2i). crude enzymeextract" f.o. *h"u, g".,n .'er" able to hydrclyz€ malathion to. dimethylpho;phorothionareanO Oimettrytptros- phorothiolthioDare (,126). Metabolism of O-etnyi O1+_methyfthioyphenyrj.-propyl_ phosphorodirhioare(sulprofos) was studied in "ouon 1f24. ih;;;., merabolires were the sulfoxide and sulfone deriyatives, indicating o,,idation usi major initial transformation. Formation of the phenol and gluociside conlugutes of sulprofos indicates hydrolysis_of rhe phospho-phenot bondl b", "";il;;i;;;;;lysis has not been confirmed. However, other enzymes, such as mixed-function oxidases and glutathione S-(GST), may Le equally imponant in itre-leto*ittcat;on of organophosphorusinsecticides in plants (.f2g).

Hydrolysis of OrganophosphateInsecticides by Microorganisms

The degradation of organophosphate insecticideshas been studied €xtensively in several gram-negative bacterial pseudomonas strains, especia'Uydrolysis y diminuta and in Flavobacterium ATCC 2.1551 (t2g). of the oiganopf,ospf,orus occurs via nucleophiticaddition ly":f]d:: ofwatei acrossthe acid'innydride bond; tnus tne_enzymes named parathion_hJdrolasesand phosphotriesterasesare actually organophosphorusacid 'the anhydrases(r30). paiathion-nyaroiasecan be either cytosolic or membrane-bound, depending upon the bacterial species. The Flavobacterium enzymd is membrane-oounaland is a single ""i, .f:j kDa, whereas the enzyme from strain sc (gram-negative, oxidase-negatiu"u"roui", non-rnotir", ,oo sha!:d b^aggliy) is composed of four identical .uU'*io, "u"f, rvi,i a molecular weight of 67 V.0J,^(129). This enzyme is also a mernbrane-Oo"nJp-"i" (t2g). A coumaphos-degrading Nocardia isotate,B_l (/3/) producesa cytoilasmrc parathion hydrolase composed of a single 43 kDa subunir tiZq;. fn"r"'r"iufr, indicate that enzymes,possessing very diverse characteristicscan have the samecatalytic function. The aryldiphosphatasegene from Nocardia stain B_l (adp-;;n;t;as nothing in common with genes opd from other sources, and t"i rno'st likeiy undergone independentevolution (t 32). The bacterial parathion-hydrolase ,. . genes (opd) have been cloned from p. atmtnuta tJ4) \tJJ, and Flavobacterium A-ICC 27551(135). The nucleotide sequencefor the Fktvobacterium p. and d.iminutaopd genesare identical (134, 135). The opd genes-were poorly expressed in E. coli, inain" f touoO,ori"rirn hydrolase was a much larger protein when expressedin E. coli compared to the native Flav.obacterium hydrolase- When thi hydrolase was "^p.".JJ in Steptomyces Iividins, it was of similar size to that produced in Flavobacteriurn, bua was synthesizedin larger quantities and was secretedextracellularly 'b""uu""(,fJ6). production of an extracellular hydrolase is ideal for remediationpr*"rr"a detoxitication does not require bacterial uptake.

--- 77 Nitrile Hydrolysis in Plants

Nitrile groups are essential moieties in the phytotoxicology of the herbicides, (3,5-dibromo-4-hydroxybenzonitrile), [ 2-[[4-chloro-6-(ethyl- amino)-I,3,5-triazin-2-yllamino]-2-methylpropanenitrilel, and dichlobenil (2,6-di- chlorobenzonit le), and the fungicide chlorothalonil (2,4,5,6-tetrachloro-1,3- benzenedicarbonitrile). Initial enzymatic hydrolysisof the nitrile group produces an amide. The amide is subsequentlyconverted to the carboxylic acid, which may be decarboxylated. This metabolic pathway occun for bromoxynil in wheat (1321 and for cyanazinein wheat, potato (Solnnum tuberosum)and maize (Zea mays L.) (138, 139).

Nitrile Hydrolysis in Microorganisms

In bacteria,the cyano group of bromoxynil can also be hydroxylatedto the respective carboxylate by several speries: Fexibacteriun sp. (140) ar.d Klebsiella pneumoniae (l4l)- The K. pneumoniae utilizes bromoxynil as a niaogen source, rather than a carbon soutce, with 3,5-dibromo-4-hydroxybenzoateaccumulating as an end-product. Alternatively, an oxidative pathway, mediated by pentachlorophenol-hydroxylase (flavin monooxygenase)from Flavobacteriumsp. ATCC 39723 (currentty classified as a Sphingomonas),directly liberatescyanide, forming dibromohydroquinone(,f42). Formation of the hydroquinone derivative, rather than the hydroxybenzoate derivative, renders bromoxynil more prone to complete mineralization. Klebsiella bromoxynil- genes (brz) have been cloned, sequenced,and the protein purified (/43). The bxz genes have been expressed in plants, resulting in bromoxynil-tolerant plants (,f44). Commercial application of this technology is currently being usedin cotton and potatoesto produceherbicide-resistant crops.

Role of Phosphatasesand Sulfatasesin PesticideDegradation

There is limited literature available on the role of phosphatasesand sulfatasesin pesticide metabolism. The insecticide endosulfan U,2,3,4,7,7-hexachlorobicyclo [2.2.1]-2-hepterc-5,6-bisoxymethylenesulfitel is metabolizedyia both oxidative and hydrolytic mechanisms in vitro by the white rot fungus, Phenerochaete chrysosporium (145). Under both nutrient-rich and nurientlimiting conditions, endosulfan is metabolized to endosulfan diol. This indicates a different metabolic route, catalyzed by a sulfataserather than a lignin peroxidase. Other studies have shown that endosulfan diol is also formed by hydrolytic cleavageof endosulfan by static cultures of the fungus Trichoderma sp. (.146). Theseobservations also suggest a role for sulfatasein fungal metabolismofendosulfan. 78

Genetic Engineering of Crops for BialaphodGlufosinate Resistance

Numerousphytotoxic ..,ubo,i":^T:lu:ed rqenrrrred by microorganismshave been and resledfor rheir porential isolated, ^ i.oi"ii"rl-.O? r"lese.uialaplos t:il' mostsuccessrur. cruro,ii"r.' ano ;ff:31il:lH:ll, ffi ,T: r-".*oniumsart or irerbicides'6u:**;tt"*;*$;d;i.;.,#,.',*:il,:f;;1f(;rit;;r.'fr:l^"t^^l"* been developedas major commercial moretyffi(o yietdrhe ry'_acetyt "ornqo::q f"-_prrrotJ"i*i]lt-.uy tr nyororasesand/or transferases royield,th" * acredon by metabotism *ti"" piy,ot.ii" ti,f;;_, Fu hermore, of rhepeptidyl "o.l:lrr3 bi"l"Fh*,-;;;;.;:"iitl'n""" fl:lf, ";;; ;;;"' ;"'""i..Tli'orkansam ransfo.,n"a l]"i.[11iff,ll? ] ffi "compr]:g__"f,l* inase Bialaphos .. . is a tripeptide lnyoroxy(methyr)phosphinyllbutyric a unique amino acid. L_2_amino_4- compound icia rpp.rl["teiio t*r'.]""ir, moieties. wasisolated uo, *,:t,=::l i:,",;!i;;;;;;;;rii"jil,",", The streptomyceshygroscopicus (l4g).^,The r,oO,^^o naturalform of pfrl. and rt was the first reported ,i..i_i.o.*

o gH_oHoH-OH tt il/ cHrl_cH2 _CH2{H_C-NH_CH_C_fH_CH_COOH|;?-' llll oH NH, tr, cH: Bialaphos o ll cltrP{HfCH2_CH_COOH II OH NH2 phosphinothricin

o tl cHrP_CH2_CH2_CIr_COOH lr oH NACO_CH, lr'_Acetyl_phosphinothricin

Figure 4. Chemical structures of bialaphos, phosphinothricin and N_acerylated phosphinothricin. 80

Biotechnological - _ approacheshave been utirized in srudieson the biochemistry of?PT in microorganisms and plants. The biosyntheticpuii*uy of Uiufupf,o.f,* be€n completely elucidated using various techniquesi/6/). Beginning with precursorscontaining three carbonatoms, bialaphos ii prju""j in a complexseries of o_vera dozensteps (,f61). one stepinuoru". "n u""tyicoa-o"p"nJ"nt ,"u"tion tt ut modifieseither demethyl_ppT ppT. or The Dcr genei, ;;p;;ril;;. ."sistancero in S. hygroscopicus, l3laphos and encodesfo, tt-" """tyt t inr?"r".",-aim.rgr, which convens non-phytoroxicmerabolire tiozj- this acetyl l];:":l- rs not^ctassjtied::ylllf- as Iil*"r":: a hydrolyticenzyme p?/ re, it do€sform an amidebond be, susceptibleto hydrotyticand/or rransferaseactivity. As pornreo::,::i". out T"-"11e,prevtously, deacetylaseshave been s(udied in plants,microorganrsms, as well.asin mammaliansystems, and nitroacetanilid€auO",.ut", t uu" U""n utilizedto facilitateassaying their activities(/63)_ Suchpf"n, o. *i".oiiui"nzymes could act on N-acetyl-PPTto releasethe phyroroxrc comDound. prrf.

jlT-tit:11o"*In t"nes impaning_resisrance ro pp[. cto"ins ;f; th.esisranl gene \oar) rfom 5. hygroscopicus(164) andthe transformation of ppf_resistantplants has beenaccomplished (16J). A similar geneQ)at) *on S. ,iriaiciro_ogenes Ti 494, with thesame function, wassimultaneously isorated and has also been introduced into variousplant species(160, 166, I6n. presently,more than 20 crop plant qpecies haye been transformed ppf for resistanceto in this manner. Some of these g.eneticallyaltered plants ppT areresistant to at ratesas high as I fg fra-r(ca. l0 times field apptication.rrte) (t64). :T^!:-::1 i"T1f rhis indicatesi high degreeof Incorporallonot acetyltransferase expression. The previouslyknown anti-fungalactivity of bialaphosand glufosinatewas pathogens(Rhizoctoiia lTj":l-i::::t1,rn .tfree sotani,'Scterotiii nomoeocarpa, in vitro and in vivo on ppT_resistanrrransgenrc ::::!:ti:-.yynl!rat!n) (Agrostis :r::pfng- 9entgrass palustris), an importantturfgnss (/6g). Results indicatedthat bialaphos can simultaneouslycontiol weedsu,ia fungutpathogens in this transgenicgrass. Furthermore,bialaphos tras antibiotic ""ii"i,y'?!ui"r, n "rlrr; Kiihn that causesrice sheathbligha (t6i), andMognoportu" griilHerben) Barr (r+d) rnat causesnce blast disease. Substantialsuppr"ssi,on of sheathblight symptomswas reported whenbialaphos was applied to transgenicplants which had R. sola.nipno.r rs herbicidetreatment (liojlli*'"i""a ou".g""i" nce5:"-',lt_:^.:9,:tln, pfa s lbrataphos-resistanr(bar) genelhad reduced lesions and otnersymproms of_,riceblast disease after bialaphoi triatment(t7t). I; i;;;;d that these by bialaphosand ppT, f,::q:?: T.::Tlit* becausethe microbeslack theabirity rhe compoundsto non-fungitoxic :"^:li1,l^i:r:-Tlize producrs.Thus, r. appears possrDreto controlsome seriousdiseases by using,ar_transgenic rice cultivarsand bialaphosfor weedand diseasecontrol. Glufosinitetu, ut.JU""n ,u"""ssfully used to controlthe weedr€d rice (a conspecificweed ofcultivated rice) in bar_transformed rice( 172\. among ,"^., _tl:]il1::,:lo.Pm.":" :ligl" commerciatherbicides in that they have ootn potentantrbrotic and herbicidal.propenies. This dualshategy will no doubtbe utilizedmore widely with theincreasing availability ofppT_.erisrint c.oo". 81 Summaryand Conclusions

Gene.ally, our understanding of microbial hydrolytic enzymes has been greatly increasedduring the past decade,but information on ptant hydrolytic enzymes is not as advanced. Although advances have been made, most of the information on microbial hydrolytic enzymes,has not been focuseddirectly on pesticidemetatrolism. Many hydrolytic enzymes have been reported to have multiple activities (amidase, esterase,transferase), but most have not been examinedfor multiplicity, especially with regard to the metabolism of pesticides. Also the knowledge atrout the-precisl physiologicalrole of thesehydrolytic enzymesis insufficient. Moreover, information is neededon enzyme mechanismsand regulation of enzymeactivity. Many enzyme active sites or receptor sites recognize only one sterochemicalgeometry. Thus, understandingenzyme multiplicity, physiological role, mechanism,and regulation, may lead to the developmentof more specific regulators(e.g., inhibitors, activators), so that more specific and efficacious pesticidalcompounds can be developed using a biorational design. The use of techniquessuch as protein engineering*uy prouid" additional insight on the relationshipofprotein sructure and substratespecificities of hydrolytic enzymesin plants and microorganisms. Many industrial synthetic processesproduce racemic mixtures, in which only one enantiomeris triologically active. Hydrolytic enzymeshave high potential value in the developmentof bioprocessesfor production ofcompounds uslfui to agriculture and other industries. Enzymes have the unique ability to facilitate stereospecifii transformationsand thus, biosynthetic approachesmay be more effective in some industrial syntheses. The cloning of an Arthrobacter eslerase g€ne that stereospecificallyproduces (+) r-chrysanthemicacid, utilized in rhe synthesis of pyrethroid insecticides,is one example demonstratingthis biotechnologicalstrategy. This enzyme occurs in low amounts in ahis bacterium,however cloning and over- expressioncould permit induss-ial-scalepreparation. Certain hydrolyric Jn.y*", -. also being considered for remediation of contaminants,e.g., nitrilases for solvents such as acetonitrile (/73), amidasesfor acrylamides(174), and [6-chloro_N_ -methylethyl)- ethyl-N-(I 1,3,5-triazine-2,4-diaminel chlorohydrolase, to degrade arazine (seechapter by Sadowskyand Wackett in this volume). As we have discussed,crop engineeringfor resistanceto herbicides,based upon microbial hydrolytic enzymes, is a commercial success for bialaohos and phosphinothricin. Future herbicide technologiesmay utilize other unique microbial hydrolytic enzymes that can be developed for engineeringcrop resistanceto other herbicides. Other novel pesticides(fungicides, insecticides and herbicides) may also be designedas potent inhibitors of hydrolytic enzymes,or that would be activated or detoxified by specific plant or microbial hydrolytic enzymes.

References

I . Veerabhadrapp4P.S.; Montgomery, M. W. phytochenistry1911, I0, I l.l1_ 1t'74. 82

Incledon,B. J.; Hall, !. C.In Reeutation.ofEnzymatic Systems Detoxifying Xenobioticsin pl{1n rs;Hauios, r.f., fa., ff "i". e"-##;;."',"ih" Netherfands,1997: pp 67_g2. 3. ' Gordberg'R Huet' r' c Pemolle ,t'li!:;i;" ; '' I- c. Phttochemistryrsss, 4. ""tt, r. c.; stephenson,G.R. perr,a physiot. ,"fffi't', Biochem 1991,4t, 5. facobso3,f.; Shimabukuro,R. H. J. Agric.Food Chen. 6. Brown, Igf}., 32,742_746. H. M.; Fuesrer,r. p.: Rav,r. d., 56*1';"];.;; Pestic.Chem.. VCH Vertagsgesen."r,"ft, )"; ini,. ,onrr. t;i;ir,'C" i^i", ,osi,',, zu- 7. Brown,H. M.; Neighbors, pesric S. M. Biochemphysiol- 19g7,29, 8. ' Huwe.r. K.: Lamoureux, pesrrc I lZ-lZO. lr:'ffi],?;.o G.L. si,i*iiirLt, luusE, 9. Crafts,A.S. J. Agric.Food Chem 1953,/, 5l_55. 10. D. J.; Rogers |o16, B. J. Weeds1960,8, 436447. ll. Crafrs,A. S. Weeds19fi,8, 19-25. 12. Bates,A N.; spencer,D. M.: Wain,R. L. Ann-App!. Biol. 13. Kfoppenburg, 1962,50, zt_32. D. J.; Hall, J. C. weed Res.tggl,30, g_2O. t4. flgon^ng-g. D. J.; Hail, J. c. weed.Res. rsx',jd. it1_ata. 15. nrke,S.o.; Kenyon, W. H.tnHerbicides: Cn":iiirt, ilir"a"tion aadMode ?{A!y::Keamev,P. c.; Kaufman,D.D., E<1.., t;;"iD:;;;:;;;r1.r"* York,NY, 1988, pp 7 r_t 16. 16. *1,t, 9' Schmidr,R.R. Weed Res. rs77, 17,233-239. 17. Hill,B. D; Stobbe, E.H.; Jones, B. L. WeedRes- 6iS, rc, l*S-tSq. t8. C.orecka.K.; Shimabukuro. R. H.;walsh, w. c. phy"t i it ri. iiij, X, Ss_ t9. Tal, A.; Romano,M. L.; Stephenson, c. R.; Schwan,A. L.; H all,!. C. pestic- Biochem.Physiot. 1993,46, tgltgs. 20. Teresiak,H.; Sbeer,J. p/oc. E. South.Weed. Sci- Soc. 1gg4,47, 2l_22. 21. Hoagland,R. E.; zablotowicz,R. M.; St *t,J.E.t; ;;;;.'t;;;;7"^*. Congr.Proc., Vol. III. 1996,8 t9-826. tiear, D.S. ln Herbicides:Chemistry, Dcgradation, Keanney,pc. andMode ofAction; andKaufman, D.D., rfus.; ri"r""l;;Lil i;;:N;* 1975;\tol.2, pp 541_6O7. "".*, *", Kdrcher,H.; Kellner,H.M.; Liitzscb,K.; Dorn,E.; Wink, proc. Prot. O. Br. Crop Conf. Weeds. 19E2,I , 341-34.t . 24. Smith,A_E; Aubin,A.!. Can.J. SoitSci. 199O,70, 343-35O. 25. G_afnor, J. D. Soil Biol. Biochem.1992, 35, Zg_32.' 26. Win-k,O.; Luley,IJ- pestic. Sci. lgga,22,3140. 27. ; Hoagtand, 30,?,:y""^!ry R.E.;Staddon, W. J.;t ocke,M.A. J. Agric. food Chem.2(XXl,In oress 28. C,ennari,M.; Vincenri, M.; Negre,M.; Ambrosoli,R. pestic Sci.1995,44,299- 83

29. Hoagland,R. E.; Zablotowicz,R.M . J. Agric-Food. Chem 199g,45,4.:i59_ 4765. 30. Choi,K. D.t Jeohn,C. H.;Rhee,J.S.; yoo, O. J.Agru'.aBiol. Chem.1990,54, 2039-204s. 31. Devries,R.P.; Michelsen, B.; poulsen,C.H., Kroon, p.A.; vanden Heuvel, R. H.; Faulds,C. B.; Williamson,G.; vanden Hombergh, J. p.; Visser,J.,Appl. Environ. M icrobio l. l9n, 6, 46384644. 32. Politino,M.; Tonzi,S. M.; Burnett,W. V.; Romancik,G.llJsher,J.!. Appl. Environ.Microbiol- 1997,63, 4BO7 4gll. 33. Nishizawa,M.; Shimazu,M. Ohakawa,H.: Kanaoka,M . Appl-Environ. Microbio l. 1995,6 t, 3208-3215. 34. McKay,D.B.; Jennings,M. p.; eodfrey,E. A.; MacRae,I. C.; Rogers,p. J.; Beachan,I. R. J. Gen.MicrobioL. 1992, t 38.7Ol_7O8. 35. Khalameyzer,V.; Fischer,I.; Bornscheuer,U. T.; Altenbuchner,J. Appl. Environ. M ic robiol. 1999,65, 477482. 36. Brenner,S. Nature 1988,334, 528-53O. 37. Cygler,M.; Grochulski,P.; Schrag,!.8. Can.J. Miuobiol.1995, 4/ (Supplt), 289-296. 38. Feng,P. C. C.; Ruff, T. C.; Wangwala,S. H.; Rao,S. R. pe.rrrc.Sct t99E. -5q 89-t03. 39. Neidert,K.; Van Epps,L.; Welch,W.pestic. Biochem. physiol. 1985,23, 221- 227. 40. Frear,D. S.; Still, c. c. P&yrochemistry 196t,7, gt3_92\. 41. Adachi,M.; Tonegawa,K.; Ueshima,T.Noyaku Seisen Gijutsu 1966, 14, 19-

42. Hoagland,R. E.; cral c. WeedSci. 1972.20.303-305. 43. Hoagland,R. E.; Graf,G.; Handel, E.D. WeedRes.1974. t4,371-374. phytochemistry 44. Hoagland,R. E.l Graf,Q. tg71, I t, 5Zt-527. 45. Hoagland,R. E. Phytochemistry1975, 14,383-386. 46. Hoagland,R. E. PlantCell pttysiot.lyIB, Ig, t0tg-1\27. 47. Smith,Jr., R. J.; Flinchum,W.T.; Searman,D. E. U.S.Dept. of Agric., Agric. HandbookNo. 497, t9t7,7Bpp. 48. Jun, D. J.; Matsunaka, S. Pestic. Biochem. physiol- lgg0, 3g, 26-33. 49. Carey, V. F. III; Hoagland,R. E.; TalbetgR.E. WeedTechnol. 1995.9. 366_ 372. 50. Carey, V. F. III; Duke, S.O.; Hoagland,R. E.; Talbert, R.E. pestic. Biocnem. P hysio L.199 5, 5 2, | 82- | 89. 51. Carey, V. F..III;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;Hoagland,R. E.;Talbef,R.E. pestic. Sci. 19W.49.333-338. 52. Leah, J.N.; Caseley,J. C.; Riches,C. R.; Valverde,B. pestic. Sci. 1994,42, 281-289. 53. Oyamanda, M.; Tanaka, T.; Takasawa, y.; Takematsu, T. J. pestic. Sci. 19g6, I I, 197-203. 54. Akatsuka,T. WeedRes. (Japan) IV19,24, 55-63. 55. Walden,A.R.; Walter, C.; and Gardner, R. C. Cene Bank prorein euery Accesion# AADOI946. 84

56. Hoagland, R. E.; blotowica-R. M.;.Locke, M. j,hiitr3's'nps".-A. ln Bioremediation Through 57.<1:!'!:;{::::#:::33'**6:13j;,1?i Wright,S. J. L.; pesrrc Maule.A. Scrl19g2, 13,253_256. pu t i'' s i' ; i ",;'i ;*ff;il:.ii."l"li,"llii?i'-l#ii'",x,1:!;::,T!,iTk:!;ii_l:,i3. 3:fi.1,-,k,T::ff;i,"#T'r h:v;t. $87. 27. | 82 -| 8 8. tt " ';tisler, H' D.;Kaufman, D. D. pesrrc physiot. ff;:**b . Biochem. !99r,39. 62. I-anzilforta. R. p.; pramer, D. App!.Microbiol. lnl, tg, 307_313. 63. Wrighr,S. J. L.; Sraintrope,A. i.: f)ou!\s,!. D. phytopathol. t2,51-.10. Acta Hung. 1977, 64. D.;.Bordeteaux, ll1Y: T .u, L. M.Appt. Microbiot. rs6s, tB, 36s_37s. 65. Hrmmond,M.; price, c. p.:scaven, M.b.Ez;.j.'si"i"rl.iitiiir"r,, urr- 66. Hoagland,R. E.; Zablotowicz, R.M. pestic. Biochem.physiol. Z(n. lrggi, 52, lg}- 67. Hirase. K.; Matsunaka.S. pestic. physiol. p. Biochem 1991, 39,"' 3O2_30g. 9l 5:i*"t, c.: rkufman,D.D. sciencersis'ija), 69. Blake. J.: ia;;i,. ; Kaufman,y: D.D. pr ,fii!,T,;!:i;,,;i?:.,,305.3,3 */ f . Luf::r4:i frngron. J. E.;7;;:,;;,u;{ffiiil walker. A. Soj/Biot. Biochim. tlil,1t, ' An-ow. ]? PTl. I ,.r,lmer,B.sciezce rs61, ts6. torz_lJri.' ::'-"- :li',n?R:r.F:1id. Food chem ts68,t6.6o2_6u. fril,i',Xi'f,;X;Ilhli,-i; n.E.; xnieht, s.s.; cash, B. /J. I;*.,*agtana, Yoshioka.H.: Nagasawa,T.: yamada,H- Eur. J. Biochem. lgg', t99, 17_24. il 3]l r'".'r:aln.E.; Krisch, K._oa,. i. ni"n"^1"ii'it.Tilrun. r r' rournand,D.; Bigev, F.; Arnaud, A. Appr.t"riror. iiioiiot. rggg,64,2g44- " M';Hoasland' R Ti#lIi;:R E ; wagner,S'c. sa it Biot-Biachem. ree8, M..Hagedorn, l? Ij"1":, M.L.J. Agric.Food Chem. 1g75,23,7.t-Bt. l? Y:.Gd:".L- L.; riedje. r. M.r-. A[ric. r*a ii"^ qta. t' r" rurco'n' r.; iiii,'ii,'qil- Iiilli xono'pta,A.App i.'n,i'il i,r"i)i,u"t.tsst, s7, 82. Martin, M.; Menes, G.; Allenr J';Alonso, AppLEnviron. Microbior.r#;irl#;Htez' R';Ferrer, E. P.S.: Behat. F.J.J. Bqcternt. ,.ll L'1.r, tglt, t08,g0g-8t6. S.:osawa. J.; Chune, M. c. flrff;: va_aaan.-iu"r.'].' aiornr^. rssz,20s, 85. Chebrou, H.; Bigey, F.: Arnaud, A.tcalzy,p. Biochem I2g8, Z8S-293 Biophys.Acta 1996, 86. Sorci, J.J.; Macalady, D.L. J.Agric. Food Chem 1993,4t, 1760_1766.

- tt 8s rurco,R. F.; J,i"iX,r1]r&J; Konopka,A.rpp t. Environ r xjtilL,;";; x,^I:_:.,:* i,::;:::. ,Micr.bi.,,ee4. - n:r:*,t:;ti,fqi;ffi\ffil ;:ii:,,*tt*,,, ;i :xiii;iffii: ;.,.lit :lliii{ :S: f;,;,,, o.. o, 94 Bowting, o"^,s7,, ts, 204. C. C.:Hudginr fig*'qu,*$Nfiff**'lrm, ,'"{ti#,Hgl#*:i=r'iJtii"-nxr,i!,,*. "'t', ".o*n,M. r. Bur.Environ. ,*,,rr,,sr*!r$riortu$uw* !ij:::\ilo'{lo"^ contam.roxicot. t -'; Beller, M.;rackso n.R. t. Arch. ,lj 1'X{[l;:' Environ.Heatth rseo, 4s. trl*i'i;t* t::::,1:a iI i,i;,!rti; :Vi ,,;##Uu1$:IiFil; i:iffi;i.?"^trt;:3 ?di;ff;?fl;,

l:gtl',-#trii^ii'#:i#:{i:,#i{::?:;i:n:ifi,:,:,s'; chaudhry, i#*tt""su, c . R.Appt. Environ. jll_tjt Microbior.ty)r, 57, 744_ inffiW:i:ii$,#:;r:!i;*t,,, *,,.^, 86

120- McClung,G.; Dick, W. A.; Karns,J.S."t Agric.Food Chem- 1994,42,2926_ 2931. l2l. Dick, W. A.; Ankumah,R.A.; McClung, C, Abou_Assaf,N.lnEnhanced Biodegradationof Pesticidesin the Environment,Racke, K. D.; Coars,J. R. Eds.;ACS Symp.Ser. No.426, Amer. Chem. Soc. Washington, DC, 1990,pp 98-1t2. 122. Nagy,I.; Compernolle,F.; Ghys,K.; Vandertyden,J.; DeMot,R. AppL Environ. Microbiol. l99S,6t , 2056-2060. f23. Shao,Z. Q.; Behki,R. M.,4ppLEnviron. Miuobiot. tggi,6I,2O6t_2O65. q.'; l?1. lh*Z. Sehki,R.M.Appl Environ.Microbiol. tgg6,62,4O3-3O7. 125. Rowlands,D. G.J. Sci.Food Agric.1965, 16,325_330. 126. Rowlands,D. G. J- Sci.Food Agric.lg(f., 17, g0_g3. 127. Blll, D. L.; Whitten,C. J.; Ivie, G. W. J. Agrjc.Food Chem. 1976,24, 601_ 605. 128. Lamoureaux,G. L.; Frear,D.S. In Xezobiotic Metabolism:In Vitro Methods, Paulson,G. D.; Frear,D. S.;Marks, E. p., Eds.;ACS Symp. Ser. No.97, Amer. Chem.Soc., Washington, DC., 1979,pp77-12g. 129. Mulbry,W.W.; Karns,J.S. Appr. Environ. Miuobiol. lgg9,55,2gg_2g3. | 3O. trwis, V. E.; Donarski,W. J.; Wild, J. R.; Raushel,F.ivI. Aiach emistm tgE|.. 27, t59t_1597. 131. Shelton,D. R.; Somich,C. J.Appl. Environ. Microbiol. 19ag,54,2566_2S.tt. 132. Mulbry,W. W. Gene,1992, 12, t49_t53. 133. McDaniet,C.S.; Harper, L.L.; Wild,,!.R.J. Bacteriol.lgtf', 170,23O6_23t1. 134. Chaudhry,G.R.; AIi, A. N.; Wheeter,W.B. Appl.Environ. Miciobiol. t9E8, 54,288_293. 135.Mulbry, W.W.; Karns, J.S../. Bacteriol.lgtg, tZt,674U6746. 136. Steiert,J. G.; Pogell,B. M.; Speedie,M. K.; Laredo,.,1.Bio/Technology 1989,7, 65-68. 137. Buckland,J.L.; Collins, R.F.; pullin, E.M. pesrrc.Sci.1973,4, 149'162. | 38. Benyon,K.I.; Stoydin,G.; Wright,A.N. pesrrc.Sci. 1972, j,2g3-3o5- 139. Benyon,K.L; Stoydin,C.; Wright,A.N. pesrrc.Sci 1n2,3, 37g-3g7. | 40. Cullimore,D. R.; Kohoup,M. Can.J. Microbiol.1y14,20, 1449_1452. l4l. McBride,K.E.; Kenny,J. W.; Stalker,D. N. Appl.Environ. Microbiol. 19E6, 52, 32s-330. 142. Topp,E.; Xun, L.; Orser,C.S. Appl-Environ. Microbiot. 1992,58,5o2_ 507.t35. 143. Stalker,D.M.; Malyj, L. B.; McBride,K. E.; "/.Biol. Chen 1988,263,63tO_ 6314. 144. Stalker,D.M.; McBride,K. E.; Malyj,L.B- Science19fx.E,242, 4tg413. 145. Kulfman,S.W.; Matsumura, F.Appl. Environ. Microbiol. 1996,62,593-6(N. 146. Katayama,A.; Matsumura,F. fn viron.Toxicol. Chen.1993, Ii,l059_1065. 147. Bayer,E.; Gugel,K. H.; Hiigele,K.; Hagenmaier,H; Jessipow,S.; Kiinig, W. A.; Zihner, H. Helv.chim. Acta 1972,55.224-239. w

T': Watanabe'H'; Totsukawa' 148."" Kondo,Y.; Shomura,T'; Ogawa'Y'; Tsuruoka' ReptMeiji SeikaKaisha 1973' 13' iit'str*t, f.; Moriva'C ; YoJida' l sci 34-44. T'; Kondo'Y'; Ogawa'Y; r+S. iiiou, r.; tnouye,S.; Tsuruoka' T: Shomura' H' Germ'Offen' DE 2 236 watanabe,H.; Seki'owu,Y ; waianube'T ; Igarashi' 599,t9'13. Germ'Offen DE 2 717 150. Rupp,W.; Finke,M'; Biennger'H'; Langelueddeke'lP' 440,l9't'1 . 'l Y'; Takematsu"f' J' Pestic'Sci' 1986' l5l. Tachibana,K.; Watanabe, '; Sekizawa' t 1,n-31. FoodChem' 1992' 40'165-168' 152. C;;*, M. A.; Stephenson,G' R' J Agric' J6' 393-397' iSi. J.r.n e'.e. L Agric' FoodChem lgES' MicrobioL19E9' 55' ?11-716' is+. s;;i'"1t"""""" K.t r"iv.,c' t'pptEnviron' rechnicalInformation Bulletin' iij. ri"*tttr'co'p iltz' Hoe-ooo6l N,IF 246/.Somerville, NJ' Duke'S O" Ed'; CRCPress: Boca rso. v*if,l.r. r" A erbicide'ResistantCrops; Raton'FL' 1996;PP 85-91' Proc''6th (t986) ln'ematL consn rs?. i;;;;;' K peiiic. sci'Biotechnot' P estic.Chem., 1987'145- I 48 e.; zi"gl e1c' z' Natu'Torsch'1989' 44c' 9'7-lo2' -- ' .^ f SS. wild, plan reE6'] 2 3'.1-3s'1 42 i ;;. ;;;:["f' n.; wito' e' r' t Phv siot' 1992'187' t42'l5l' iiO. i*t".'* , Broer't ; Piihler'A Planta - A'; Nagaska'K'; Thompson'C"/' l6l. Murakami,T.; enzai, fr'; tmai' S; Satoh' Mol. Gen.Genet' l9f!6,205 ' 42-50' T ; Har-a'O'; Itoh' R'; Imai' S'; 162.'"- Kumada,Y.; Anzai'H'; Tattano'C ; Murakami' 41'1839-1845' i^to-tt].t N" goaka,K' l Antibiot'1988' l91l'41' 313-319' f ot. it"gf'r"d. n.-e':Ciat'G' Enzvnologia R':Crameri' R l Davies'J E i 164.Thompson. C. J.:Movva, N R;Tig"ard' lg87' 6'2519-2523' Lauwereys'S. M'; Botterman'J' eUAO l 1l65. M; Dockz'J'; Thoen'C'; Gossele' DeBlock,M.; gott"t^un' t'; V;nderwiele' J'; l'eemans' l' EMBOJ' V.; Movva,N. R.;Thompson,C ; vanMontagu' 1987,6,2511-2518- Proc' BraunschweigSymp' I Amold, W'; Wohlleben'W : Piihler'A ' ln 166. Broer, ; 1989;p 240' AppliedPlant Molecub' aiit" Caling:Braunschweig;^Germany' D'; Strauch'E': Piihler'A' r ol. #5iii"u."l w.; Arnold'w : Broer'l lHillemann' Genel9E8' 70,25-3'1' D'; Sticklen'M' WeedSci' 199.8' 46' 168. Liu, C. A.; zlong, H ; Vargas'J': Penner' r39-146. of RiceProduction: Wiley 169. DeDatta,S. K. Principlesand Practices Intemarional:New York' NY' l98l P K;Takamasu' S ;Ooba' Uchimiya,H.; I*utu' f'l', Noji'i' i l Samarajeewa' 170. Bio/Technology1993' S.; Anzai,H.; christensen,i'H'; Quait'Pti"'Toki'S' , /, 835-836. Molec'Pl'ant- H Norita'E ; Uchimiya'H'; NakamJr.a'l' l7l. Tada,T.; Kanzaki, ; -l M icrobe Interact' 1996' 9' 62--164' 88

172. Sankula,S.; Braverman,M. P.; Jodari,F.; Linscombe,S. D.; Oard, J. H. I,/eed Technol. 1997. I I.70-75. 173. Babu, G. R.; Wolfram, J. H.;Marian, J. M.; Chapatwala,K.D. Appl. Microbiol. B iote c hno l. 1995, 43, 7 39-7 45. 174. Nawaz, M. S.; Khan, A. A.; Seng,J. E.; kakey, J. E.; Siitonen,P. H.; Cemiglia, C. E. Appl. Environ. Microbiol. 1994,60,3343-3348.

ReFintedfrom ACS Symposium Series 777 PesticideBiotransformation in Plana dd Microorganisrns Simil*ities andDivergences J. ChristopherHall, RobenE. Hoaglad,and Robert M. Zablotowicz,Editors Published2001 by AmericanChemical Society