Chapter 4

The Rote of Plant and Microbial HydrolyticEnzymes in Pesticide Metabolism

Robert E. Hoagland and Robert M. Zablotowicz

Southern Weed Science Research Unit, Agricultural ResearchService, U.S. Department of Agriculture, Sloneville, MS 38776

Many pesticide molecules containing amide or carbamate bonds, or

esters with carbonyl, phosphoryl, and thionyl linkages, are subject to

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 biodegradation in the environment. Substrate specificity

of esterases and amidases varies dramatically among species and

biotypes of plants and microorganisms. The constitutive or

induJible nature of these enzymes, as well as production of

isozymes, is also important in the expression of these mechanisms'

For example,'increased aryl acylamidase activity has been reported

as the mechanism of evolved resistance to the hedicide Propanil in

two Echinochloq weed species fiunglerice, E. colona (L.) Link;

barnyardgrass, E. crus-Balli (L.) Beauv.l. Other acylamidases, such

as a linuron-inducible enzyme produced by Bacillus sphaericus'

have broad substrate specificities including action on acylanilide'

phenylcarbamate, and substituted phenylurea herbicides. Many

rnicrobial hydrolytic enzymes are 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

mechanisms in plants and biosynthetic and/or degradative enzymes

in microorsanisms.

O 2001 American Chemical Society

59

Hydrolytic enzymes catalyze the creavage of certain chemical bonds of a substrate bythe addition of the components of water (H or OH) to each of the frooucts. Manyherbicides, fungicides and insecticides contain moieties t".g., ;ij" or carbamatebonds, or ".rrT yilh carbonyl, phosphoryl and thionyl finU"geO tfrat are subject toenzymatic hydrolysis. A wide array of hydrolases (amidies, esrerases, Iipases,nitrilases, peptidases, phosphatases, etc.), with broad and narrow subshatespecificities are present in animals, microorganisms and plants. plants andmicroorganisms should not n€cessarily be expected to possess the enzymatrc capacityto metabolize xenobiotic compounds. But indeed, ii is ttre muttiplicity of certainenzymes and their broad substrate specificities that make pesti;ide degradarionpossible. Thus the potential for hydrolytic cleavage of a given pesticide exists innumerous organlsms.

Various levels of cellular companmentalizarion for these hydrolytic enzymesexist. Some are cytosolic, while others are associated with membrines, mrcrosomes,and other o-rganelles. Some fungi and bacteria also excrete hydrolytic enzymes thatact extracellularly on substrates, and thus pesticide detoxification and degradationmay occur without microbial uptake of the compound, Certain hydrolases areconstitutive, while others are inducible. The abiliiy of an organism to hydrolyzecertain pesticides can render the organism resistant io that com;ouod. Many plantsare insensitive to various classes of pesticides due to their unique hydrolyticcapabilities. Furthermore, differential metabolism is an important

'mechanism in

determining _tlle selective toxicity of a given compound among plants and other

organisms. Since molecular oxygen is not involvea in hydrolyticlctivity, hydrolysiscan occw under anaerobic and/or aerobic conditions.

In this chapter we examine the hydrolytic transformations of a variety ofpesticide_ chemical classes by plants and microorganisms- The role of these enzymesin pesticide activation, detoxification and degradition is discussed, and opponunitiesfor exploiting novel hydrolytic rransformation; ofxenobiotics are examined.

Esters are susceptible to hydrolysis by esterases, and to some extent by lipases andproteases. Esterases are ubiquitous in living organisms, and occur as multipleisozymes with yarying substrate specificities and- catalytic rates, For example,fourteen esterases have been isolated from bean (phaseoius vulgaris L.) and sevenfrom,pea (Pisum sativun L.) (,f). The biochemistry and rcle o'f plant esterases inxenobiotic metabolism has recently been reviewed 1Z;. fl" phyiiological role ofthes€ 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 degrees of specificity and kinetic rates areobserved among plant esterases. For example, un """tyl "",erur" from mung bean

Ester Hydrolysis in Plants

(Vigw.radian) hypocotyls hydrolyzed high molecular weight pectin esters (primaryphysiological substrates), but could more rapidly hydrolyzJlow molecular substratessuch as triactin, and p-nitrophenyl aceta(e (3).

60

Many pesticides are applied as carboxylic acid esters (Figure l). Since the acidform is generally the active agent, esterases can play a role in pesticide activation,detoxification, and selectivity in plants (Table I.). Many herbicides have beenspecifically developed as esters to improve absorption into plant tissue, and to alter

Table L Role of Plant Esterases in Herbicidal Activitv in Plants

Mechanism Herbicid.e Plant species CitationActivation

Detoxification

Increased absorptionand translocationIncreased absorption

Fenoxaprop-ethyl

Diclofop-methyl

Thifensulfuron-methyl

Chlorimuron-ethyl

Quinclorac esters

2,4-D-Butoxyethylester

Wheat, Barley,Crabgrass

Wheat, Oat, WildOat

Soybeans

SoybeansSpurge

Bean

(4)

(5)

(6)

o(8)

(e)

phytotoxic selectivity. Aliphatic derivatives of the broadleaf herbicide 2,4-D l(2,4-dichlorophenoxy)acetic acidl are widely used. The polar forms of 2,4-D are readilytaken up by roots, while long-chain non-polar ester forms (butoxyethyl, and isooctyleste6) are more readily absorbed by foliage. The ability to hydrolyze these 2,4-Desters is widely distributed in sensitive plants such as cucumber (Cucumis sativus L.)(/0) and tolerant plants such as barley (Hordeum vulgare L.) (,1,1). In a fungicidedevelopment program, ester derivatives of the herbicide 4,6-dinitro-o-cresol (DNOC)were assessed as fungicides, and for potential phytotoxicity on broadbean (Vicia faba)(,12). Short-chain aliphatic esters (acetate, propionate and isobutyrates) were readilyhydrolyzed and were highly phytotoxic. Howev€r, aromatic esters of DNOC (chloro-and nibobenzoates) were hydrolyzed slowly and exhibited low phytotoxicity. Theuptake of clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) ftee acid was greatercompared to that of the l-decyl and 2-ethylhexyl esters in Canada thistle (Cirsizzamense) alnd wild buckwheat (Polygonum convolvulus\, and in isolated cuticles ofEuonymus fortunei (13, 14 )- De-estedfication was essential for the ldecyl and 2-ethylhexyl es0ers of this herbicide to enter the phloem and translocate to site of action.

The polycyclic alkanoic acid herbicides (PCAs), usually contain more than onering structue (one is usually a phenyl ring) attached to an asymmetric, non-carbonylcarbon of an alkanoic acid (,15). Common herbicides in this class include: diclofop-methyl [methyl ester of (a)-2-[4-(2,4-dichlorophenoxy)phenoxylpropanoic acid],fenoxaprop-ethyl fethyl ester of (l)-2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxyl-propanoic acid ), and fluazifop-butyl { (R)-2-[4-[[s-(trifluoromethyl)-2-pyridinyl]-oxylphenoxylpropanoic acid). In plants, PcA-ester hydrolysis, yielding the parent

61

H.OH

IO{II2-CO4-CH24H2-O-CH2-CH2€H2-CH3

I

2,4-D butoryethyl ester

cl/ H-oH

V.' -\__/- "-V- o-cH-co-o-cH3

Diclofop-methyl cHs

H-OH

62

acid, is the first enzymatic action on these compounds (15). For the PCAS, de-esterification is a bioactivation, not a detoxification mechanism as demonstrated fordiclofop-methyl (5). The carboxyesterase responsible for de-esterification of the PCAherbicide chlorfenprop-methyt [methyl 2-chloro-3{4-chlorophenyl)propionate] hasbeen 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 subsequent phenolic conjugation (,18), andfenoxaprop, via glutathione conjugation (.19). Rice is generally tolerant tofenoxaprop-ethyl, but under low light intensity, rice can be damaged by fenoxaprop-ethyl treatment (20). However, similar rates of in vitro a.l,d in vivo fenoxaprop-ethylde-esGrification were found in rice seedlings grown in either, normal light or low lightconditions (2,1). Esterase activity was also measured using fluorescein diacetate(FDA) as a substrate (21). In fenoxaprop-ethyl-treated rice, FDA esterase activity was4l Vo lower in shaded plants compared to unshaded plants. However, in untreatedrice, FDA esterase activity was 22Vo lower in shaded versus unshaded plants. Hence,the phytotoxic compounds fenoxaprop-ethyl and fenoxaprop acid, persisted longer inshaded plants, which may explain this phytoaoxicty to plants under low lightconditions.

The herbicide chloramben (3-amino-2,5-dichlorobenzoic acid) was rapidlymetabolized to an N-glucoside in resistant plant species, while sensitive plans formedthe carboxy-glucose ester (22). The glucose ester is unstable in vivo due to hydrolysisby esterases, thus an equilibrium between the ester and free acid is maintained.

The selectivity of the sulfonylurea herbicides is based on several detoxificationpathways, including oxidative and hydrolytic mechanisms (6). Soybean (Glycine maxMerr.), can de-esteriS thifensulfuron-methyl { methyl 3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminolcarbonyllaminolsulfonyll-2{hiophenecarboxylate } to thefiee 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-yllaminolsulfonyubenzoatel which injures the crop plant. Soybean esterasescan hydrolyze thiophene O-carboxymethyl esters and O-phenyl€thyl este6, but not O-phenyl methyl esters of certain sulfonylureas (6). Chlorimuron-ethyl { ethyl 2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)aminolcarbonyllaminolsulfonyll-benzoate l detoxif-icaction occuns via de-esterification of the ethyl group and dechlorination viahomoglutathione conjugation in soybean (Z). However, conjugation occurs three-foldmore rapidly than de-esterification.

A recent study compared the phytotoxicity of thirteen ester dedvatives (C5 toCl6) of the herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) forphytotoxicity against leafy spurge (Euphorbia esuls L.) (8). Foliar application ofquinclorac caused rapid death, whereas quinclorac esters applied at higherconcentxations to foliage caused only phytotoxicity, not mortality. When applied tosoil, quinclorac esters were metabolized by a series of and oxidations while hydrolysiswas limited. This resulted in the slow release of quinclorac which increased herbicideefficacy against leafy spurge plans.

63

Ester Hydrolysis in Microorganisms

Studies of fenoxaprop-ethyl (23, 24) and diclofop-methyl (25) degradation in soilsdemonstrated a rapid hydrolysis of both compounds to their parent acids. Thishydrolysis was more rapid in moist and non-sterile soils compared to dry or sterilesoils, which suggested microbial degradation. The role of enzymatic hydrolysis ofdiclofop-methyl was reduced with certain microbial inhibitors (propylene oxide andsodium azide) (25). During hydrolysis of diclofop-methyl and fenoxaprop-ethyl insoil, enantiomeric inversion was observed (2O. The S enantiomers of bothcompounds underwent a more rapid inversion than the R enantiomers, and rates ofinversion were dependenf on soil type. No inversion was observed with entantiomersof 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 alsoaffecs the degradation pathway of fenoxaprop-ethyl. Under acidic conditions, therate of de-esterification was signifrcantly lower than under neutral soil conditions,however, the benzoxazolyl-oxy-phenoxy ether linkage of fenoxaprop-ethyl was proneto non-enzymatic cleavage under acidic conditions (22). De-esterification offenoxaprop-ethyl occurs readily in mixed microbial cultures (28) and in pure culturesand enzyme extracts of bacteria, especially fluorescent pseudomonads (27, 29).Fenoxaprop-ethyl de-esterification in bacterial enzyme preparations is pH sensitive,with the highest activity in the neutral to slightly alkaline range. The samePseudomonas strains that hydrolyzed fenoxaprop-ethyl were unable to de-esterifychlorimuron-ethyl (R.M. Zablotowicz, unpublished results). Four distinct types ofesterases are found in a P. fluorescens strain (30). These P. fluorescenJ esterasesdiffer in substrate specificity, cellular location and structure.

Esterases have been cloned, and proteins have been sequenced from severalmicroorganisms, e.9., two fgrulic acid eslerases from Aspergillus tubingensis (3l), acephalosporin esterase from the yeast Rhodosporid.ium toruloides (32\, achrysanthemic acid esterase frcm Anhrobacter globiformis (33), and several otheresterases from Pseud.omonas fluorescezr strains (30, 34, 35). The ability of these€sterases to hydrolyze ester linkages of pesticides has not been examined. Sincemolecular oxygen is not involved, enzymatic hydrolysis can occur under anaerobicand aerobic conditions.

The active site of prokaryotic and eukaryotic est€ras€s contains the sedne motif(Gly-X-Ser-X-Gly), originally characterized in serine hydrolase (3O. This conservedpeptid€ is part of a secondary structure of the enzyme molecule located between a -strand and an a-helix (32). A general mechanism has been proposed for the catalyricactivity of the esterase superfamily (Figure 2). This mechanism involves the formationtwo tetrahedral intermediates (.t/), with the active serine serving as a nucleophileenabling ester bond cleavage. A histidine residue in a p-srand, is also involved information of an ionic attachment at the carbonyl oxygen atom of the substrate duringcatalysis. Reaction of the second intermediate, with a molecule of water,concomitantly releases the parent acid of the subsrate and regenemtes the activeserine of the enzyme. A similar reaction mechanism is postulated for the serine

64

proteases. However, a chrysanthemic acid esterase from Az& robacter sp. is similar tomany bacterial amidases that possess the Ser-X-X-Lys motif in the active site. Thisesterase may provide a unique opportunitiy for biotechnological synthesis, since itstereoselectively produces (+)-r-chrysanthemic acid, used in pyrethroid insecticidesynthesis.

Certain esterases, e.g., the R. torulaides cephalosporin esterase, also haveacetylating actiyity when suitable acetyl donors are present (-t2). The R. toruloidescephalosporin 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 animportant role for glycosylation in stabilizing the natiye protein structure.

Feng et al. (38) transformed tobacco (Nicotiano tabacum L.) and tomato(Lycopersicon esculentum L-) with genes encoding for rabbit liver esterase 3 (RLE3).This esterase gene expressed in th€se plants, confened resistance to the herbicidethiazopyr Imethyl 2-(difluoromethyt)-5-(4,5-dihydro-2+hiazolyl)_4_(2-methylpropyl)-6-(trifluoromethyl)-3-pyridinecarboxylatel. These researchers proposed, that a criticalassessment of microbial esterases may provide other hydrolytic enzymes that areuseful in conferring pesticide (herbicide) resistance in plants. Such enzymes couldalso play a role in reducing the levels of pesticides, their metabolites, and otherpotentially harmful xenobiotics in food products.

Inhibition of Esterases in Plants and Microorganisms

The effects of two fungicides, capran lN-(trichloromethylthio)_4_cyclohexene_ I, | _dicarboximidel and folpet [N-(trichloromethylrhio)phrhalimide], and a suftrydrylbinding inhibitor, perchloromethylmercaptan, were assessed on esterase activity ofPenicillium duponti (J9). Esterase activity using p-nitrophenylpropionate as subsratewas inhibited 50Vo t:y all three compounds at 0.5 to 2.0 uM. But, concentrations thattotally inhibited p-nitrophenylpropionate esterase activity had no effect on c_naphthylacetate esterase activity. The extreme sensitivity of certain fungal esterases to thesetwo fungicides, suggesrs thar part of their toxicity to fungi may be due to esteraseinhibition (39).

Amide Hydrotysis in plants

Amide and substituied amide bonds are present in several classes of Desticides, i.e..acylanilides; alachlor [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 ( 2,4 -d imethyt-Lhien-3-yl ] ; metaiaxyl l/V-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine merhyl esterl; metolachlor [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

examples ofthese substituted amides are given for comparison (Figure 3.)

Rice (Oryza sativa L.) is tolerant to the acylanilide herbicide propanil, due to

the presence of high levets of aryt acylamidase (EC 3.5.1.a), which hydrolyzes the

amide bond to form 3,4-dichloroaniline (DCA) and propionic acid (40' 4l). 'fhis

enzyme activity is the biochemical basis of propanil selectivity in the control of

barnyardgrass lEchinochloa crus-galli (L.) Beauv.l in rice. Barnyardgrass tissue was

unable to detoxify absorbed propanil due to very low enzymatic activity; rice leaves

contained sixty-fold higher aryl acylamidase activity than barnyardgrass leaves (4O).

Propanil aryl acylamidase activity is also widely distribuied in other crop plants and

weeds (42,43\. Plant aryl acylamidases have 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

serious conspecific weed pest in cultivated rice fields in the southern U.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 intensive use of propanil in Arkansas rice production over a thirty-five year

period, bamyardgrass (initially controlled by propanil), has evolved resistance to this

herbicide, and this biotype is currently a serious problem (49). Populations'of

propanil-resistant barnyardgrass have been verified in all southern U.S. states that 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 acytamidase activity (50,5/). Increased propanil 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 Product,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 metabolized futher in this species (53).

Initial assessment of substrate chemical sEuctwe and aryl acylamidase activity

has been studied in enzyme preparations from various ptants, e.g., rice (40)' tulip (44)'

dandelion (45), and red rice (46). Nine mono- and dichloro-analogs of 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-dichloropropionanilide compared to propanil;

*hil" in tutip, equal activity was observed with propanil, 2,4-dichloropropionanilide'

and 4-chloropropionanilide as subskates. In a similar fashion, the effects of alkyl

chain length on 3,4-dichloroanilide subsaates were evaluated. Propanil was the best

substrate for all four enzyme prepantions. Reducing the chain length to one carbon

66

ol l

R-O-C-R* ----------------

o-I

R-O-C-R. TetrahedralIntermediate I+ r-ort

Ewvu,Ser/

ol l

" , t ,^ *JO\ot l

/.- O_C_R+ + R_OH

f,,ra,ran Ser/ lllrO7

OHR.-C-O- I<--+ -oH /-o-!-*- #ffi#:"

Ev,rwser ,/ E"raa.rSer/ 6-

Figure 2- Scheruttic mechanism of esterase-mediated hydrolysis-

H OI l

N-C-OCH(CHt2

/N"ll lPropham\/H O

| l l

T-N(cHr,H.OII

Linuron

O Hi l l

O Hi l lctl3o{-N\f\./,, tT>ay.",

ll I Ho-H ll I\/ \/

Phenmedipham

Figure 3. Selected pestici.des with qmide and substituted amide bonds.

67

(i.e., the acetamide analog), decreased activity by 18 to 50%, compared to propanil'

i""."^i"g the atkyl chain-length or inserting alkyl branching' reduced activity by 60

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

*eights of about 150 kDa, and are membrane bound' These enzymes share a comnon

fff "op,i-utn

of 7.0, similar Km's for propanit' and are both inhibited bv the

ins""ti"ia" carbaryl. A gene for aryl acylamidase has been cloned from Monterey

pine (Pinzs radiata) male pine cones (55). This protein, comprised of 319 amino

acids, is similar to esterases containing a serine hydrolase motif in the active site'

Amide Hydrolysis in Microorganisms

As we have reviewed (56), aryl acylamidases have been characterized in diverse

species ot atgae, bacteria and fungi, and propanil has been the most studied pesticide

.uU"au,". itt" distribution of aryl acylamidases and related hydrolytic enzymes

produced by selected microorganisms are summarized (fable II)' Differences in

Iubstrate specificity and inducibility of these enzymes are found- among genera and

species of'microbes. For example, aryl acylamidases produced.by.P' fluorescenssirains RA2 and RB4 had a substrate .ung".ii^it"a to certain acylanilides (propanil'

nitroacetanilide, and acetanilide), and were ineffective on several herbicides

contuining substituted amide bonds: phenylureas (linr'non --and diuron)' a

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-i,r -Jirr.,fryi-i-p-pynyl)benzamidel ) (60. Similar substrate -specificities

are found

in oitr". U*t".iut slains (fable IU)- Other microbial acylamidases also have activity

restrict€d bo certain acylanilidis, e.g., Fz sarium orysporum (69)'

Some acylamidaies, such as a linuron-inducible enzyme produced by Bacillus

,pno"riir, (5i), and an extracellular coryneform aryl acylamidase (60)' have wide

r'uUrt ut" tp""lfr"ities including hydrolytic action on acylanilide' phenylcarbamate'

and substituted phenylurea pesticides. ln a Fusarium orysparum strain' there are two

distinct aryl acylamidases: one induced by propanil, the second induced by p-

"t foroptr"nyf methyl carbamate (69). One study has compared-the metabolism of

""u"rui ptt"nytu."u herbicides by bacteria and fungi (70)' Nine fungal species were

,no." "if""tiu" in degrading linuron or isoproturon [3-(4-isoPropylphenyl)-l ' l-

Jimethylureal "o*purJd to fenuron (l,l-dimethyl-N-phenylurea)' -but N or O-

a""itvl'",." i"., ttyirolysis) was the major degradation mechanism'- Only one of five

bacterial isolates (a psiudomonad) *"tuUotit"O linuron with the formation of 3'4-

dichloroaniline, indicating hydrolytic cleavage of the urea bond - The hydrolytic

,n""t unir. for phenylurJa herbicides by a ioryneform-like bacteria has also been

showr with un organis- capuble of hydrolyzing linuron > diuton >-monolinuron [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 nidulansAspergillus nidulans

Bacillus sphaericusCoryneformJike,strain A-l

Fusariun oxysporum

Fusarium solani

Nostoc entophytumPenicillium sp.

Pseudomonas

fluorescens

P. Jluorescens

P. pickettiP. striata

CIPC,IPCPropanil,

. (propionanilide**)Linuron, carboxin, IPC

CIPC, linuron,naproanilide,

propanilPropanil, CIPC,

(acetani I ide)Propanil, (acetanilide)

PropanilKarsil, propanil,

(acetanilide)(Acetanilide),

(p-Nitloacetanilide,p-Hydroxyacetanilide)Propanil, (acetanilide,

nirroac€tanilide),Propanil

cIPc, IPC,

nt*Constitutive

Linuron(Acetanilide)

Propanil andphenylureasPropanil and(acetanilide)

nlKarsil

(Acetanilide)

Constitutive

Consti tutivecIPc

6n(58)

(5e)(60')

(6t)

(62)

(63)(64)

(65)

(66)

(6D(68)

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, 3770 of the soil and water isolates exhibited

propanit hyirolytic activity. Although activity was observed in both gram-positive and

gram-negative isolates, there was a greater frequency of propanil-hydrolysis among

lram-negative bacteria. The hydrolytic activity of cell-free extracts of several of these

isolates, and other rhizobacterial cultues on several substrates, was assessed using

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

NOTE: *nt = not tested; ** compounds in par€nthesis are not used as pesticides'

69

E E E E E E E E E E

9 = v Y = Y = = r -

q > & e & & & / < r ' a

- 6 _ c E9 e I

q A ' c

E * Ae E 5; a : €E 6 < :t J < o -

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9p: 3 3- E S ' E: ; F E

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! \ E > , 4\ J { = ! -

E E i E6 r 5 :e ; ! ii F ; P

9 a * ae g < &- ! t r , <. F o < 9; 9 + Y

i i . ; . = ;i i - $

€ E : ?; ; " - . :. .11 E- I5 > 2 t r

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i . : I4 : : l - : ;

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E]

- ^ - 9 o 3 - , r , o . +* * = 3 : * ; : ! :

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t$tJ*Dss$d

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=- k: *s:= n i

E

z

p E s F ! [- 3 3 U 3 3 Fi e s S F F nx s 6 0 0 C ) 0i i ' s s I s i \e * 5 - : - \ i r - : :

{ a . i d q . d Q

^

a ' t

,sil *

at)

F

OJa

(a

F

70

Table IV. Recovery of Propanil-Hydrotyzing Isola&s from a Mississippi DeltaRice Soil and Flood Wattr

Source Gram'stainreactLon

Propanil- Tonl Isolatestestedisolates

1982 NegativePositiveNegativePositiveNegativePositiveNegativePositive

I24o

334

I J

t 98l 5ol l8l4

Water

1983 Soit

Water

Total soilTotal, water

l 9t'l

52

Total $am-Positive 15 59

Total gram-negative 2l 38

NOTE: Bacteria were isolated on tryptic soy agu (lo%)' from soil and warcr following

propanil application. Individual colonies werJ subcuhured, ascertained fo! purity and tested

io. b.". ,iuin ,"""tion. Propanil hydrolysis assessed in cell susp€nsions (log I l 0 cells ml; 50

-M pot^"iu. phosphate, ptl S-o; S0O llvl propanil) after 24 h incubation' Propanil and

metatolites were determined by HPLC as described elsewhete (66)'

exposed to propanil. P. Jluorescens strains RA-2 and RB4 exhibited aryl

acylamidase activity several orders of magnitude higher than any other organlsm

isolated in our studies (56,66).

The ability to hydrolyze propanil was studied in fifty-four isolates of fluorescent

pseudomonadsiollected f;omihree MississiPpi Delta lakes (74)'- Overall' about 607o

of the irolate" hydrolyzed propanil, and all the propanil-hydrolyzing bacteria were

identified u" P. luor"tr"ni biotype II. Most propanil-hydrolyzitg P' fluorescens

iso la tes t rans formedDcAto3,4 .d ich lo roacetan i l ide 'Thepoten t ia l fo race t ftransferase activity by these P, fluorescens aryl acylamidases should not be

overlooked, since the enzyme iiolated from Nocardia globerula (75\ and

Pseudononas lcidovarans (76) possesses this activity The amidase from

Rhodococcus sp. strain R3l2 has both amidase and acyl transferase activity This

enzyme was expressed in Escherichia coli and utrlize'd in kinetic studies on acyl

transferase aci;ity gn. This purified enzyme catalyzed acyl transfer fiom amides

and hydroxamic acids to onty water or hydroxylamine' Funher aspects of acetyl

tranrfe.""" activity will be addressed in the later presentation on bialaphos and

phosphinothricin detoxifi cationhesistance.' i variety of methods (radiological, sPectophotometdc, and HPLC) are available

to ,n"*ur" "n"yrnutic hydrolysis oi amides. A colorimetric method for measuring aryl

acylamidase iivity in soil using 2-nitroacetanilide (2-NAA) *,t:qt:.ut *T:":ltll

7l

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

RhizosPhere Bacteria

Genera C Recovered in methanolic extracts

DCA 3,4-DCAA

Bacillus sp.Flavobacteriumsp.P. cepaciaP. fluorescensP. fluorescensP. fluorescensP. fluorescensP. fluorescensRhodococcussp.Rhodococcus

S9282w92B14

AMMDRA-2RB.3RB-4UA540w92812S92A1

s93A2

7 4.5nd2.5nd100'70.3

15.9

nd

' 1 1 . 1

88.6

6.91009 1 . 0r00nd9.2to.2

96.r

Sw

RRRRRwS

10.5 18.54.5 6.9

ndnd

18.6Nd5.0NdNd4.23.5

3.9

NdNdt .5NdNd16.3Nd

Nd

NOTE: DCA = 3,4-dichloroaniline; 3,4-DCAA = 3'4-dichloroac€tanilide; Origin = highly

polar metabolites limmobile in benzene: acetone solvent): S = soil isolate; w= water isolate: R

= rhizosphere isolate; nd = none detected. cell suspensions [48 h tryptic soy broth cultures, log

r iO ""rL rnr't; potassium phosphate buffer (50 mM, pH 8'0)l were treated with propanil (800

trM, s.0 kBq mi-') and iniuuaied at 28"C, l5orpm,24h Propanil and metabolites identified

by TLC and radiological scanning as described elsewhere (56)'

hydrolytic enzymes such as alkaline phosphaase (3 to 5%)' and aryl sulfatase (5 to

t\vo'..' tryt acylamidase aciivity was 1.3- to 2.5-fold higher in surface no-till soils

comoared io conventional-tilled soils. This would be expected due to the greater

microbial populations and diversity associated with the accumulation of soil organic

matter in tirelu.face of no-till soils. Maximal aryl acylamidase activity 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.to chloroacetamide herbicides' there is only limited evidence in the

literature for cleavage of the substituted amide bond The fungus Chaetonium

globosum was show-n to hydrolyze substituted amide bonds of alachlor (79) and

iretolachlor (80). A unique cleavage of propachlor [2-chloro-N-(l -methylethyl)-N-

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 iLs dist bution

among other species. Recently, two bacteria (Pseudomonas and Acientobacter)

capabl of metabolizing ptopuihlot were described (82) Both strains initially

de'halogenated propachlor io N-isopropylacetanilide. "fhe Acientobacter strairj' then

hydrolized the'amide bond, befori thi release of isopropylamine, and prior to ring

"i"urug". T1r- Pseudomonas sfiain initially transformed propachlor via N-

'1,

dealkylation. This metabolite was then cleaved at the b€nzyl C-N bond as in theMoraxelle isolate described above (8/).

Aryl acylamidases have 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 enzymeshave been shown to be quite diverse. Among these four genera, the aryl acylamidasesranged in size from 52.5 kJ)^ for the P. Jluorescens enzyme, to about 127 kDa for thecoryneform and, N. globerula aryl acylamidases. The P. pickettii enzyme is ahomodimer, while the other euymes are monomers. A novel amidase from P. putida,specific for hydrolyzing N-acetyl arylalkylamines, was purified to homogeneity (84).This protein (MW =150 kDa) is a tetramer of four identical subunits. This enzymehydrolyzed various rV-acetyl arylalkylamines containing a benzene or indole ring, andacetic acid arylalkyl esters, but not acetanilide derivatives. To our knowledge, nogenes for specific pesticide-hydrolyzing alyl acylamidases have been cloned. Amultiple alignment and cluster analysis has been performed on amino acid sequencesof 2l amidases or amidohydrolases (85). A hydrophobic conserved motif [Cly-Gly-Ser-Ser (amidase signature)l has been identified which may be impofiant in bindingand catalysis. Amidases from prokaryotic organisms also have a conserved C-terminal end, not found in eukaryotes. These studies also indicate similarities ofamino acid sequences among amidases, nitrilases and ureases.

Metals, i.e., Hg*, Cu* Cd*and Ag*, that affect sulftrydryl groups differentiallyinhibited 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 thecoryneform-like aryl acylamidase by about 90% (60). An aryl acylamidase from P.aeuriginosa required Mn* or Mg* (83), but these cations were not required by otherenzymes mentioned aboYe.

Initiaf studies ofrhe interaction of chemical structure and microbial (Penicilliumsp.) aryl acylamidase activity were evaluated in an enzyme inducible by karsil [N-(3,4-dichlorophenyl)-2-methylpentanamidel (64). Activity was greater with longer alkylamide 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), compared to 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 detectedwith diuron and CIPC. The hydrolysis of seven para-substituted acetanilides by threebacterial species (Arthrobacter sp., Bacillus sp., and Pseudomonas sp.) was comparedto rate constants for alkaline-mediated hydrolysis (86). In these studies, parasubstitution did not affect acetanilide hydrolysis by resting cells of these bacteria,however alkaline-mediated hydrolysis was highly affe.ted. These studies suggestdifferent intermediates and mechanisms for biotic versus abiotic transformations.

A structure-activity study evaluated acylanilide herbicide chemical structureusing model substrates and four bacterial strains [two Arthrobacter spp. (BCL andMAB2), a Corynebacterium sp. (DAK12), and an Acinetobacter sp. (DVl)l capableof growth on acetanilide (82). When the nitrogen of acetanilide was alkylated (methylor ethyl), the compound was an unsuitable subsnate for all four strains. When a para-

73

methyl group was present on the acetanilide ring, activity occurred in all strains, butwas 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 tothat on acetanilide occuned in two strains (DAKI2 and BCL), but was only about3OEo of rhe acetanilide rate in MAB2, and undetectable in DVl. Little or no activiryin all four shains was found with dimethyl substitution in the 2 and 6 positions.Consequently, alachlor and metolachlor (with 2 and 6 alkyl substitutents on the ring)are mor€ persistent in soil compared to propachlor with an unsubstituted aniline ring(88).

The fungicide ipridione [3-(3,5-dichlorophenyl)-l{-isopropyl-2,4-dioxoimidazo-lidine- I -carboxamidel undergoes several potential amide hydrolytic .eactions.Ipridione degradation by an Anhrobacterlike strain (89) and three pseudomonads (P.fluorescens, P. paucimobilis, and Pseudomonas sp.) has been reported (90). Initialcleavage of ipridione forms lr'-(3,5-dichlorophenyl)-2,4-dioximidazoline andisopropylamine- The imidazolidine ring is cleaved, forming (3,5-dichloro-phenylurea)acetic acid, which can be further hydrolyzed to 3,5-dichloroaniline. 3,5-Dichloroaniline is a major metabolite observed in soils where microflora have adaptedthe ability for enhanced ipridione degradation (91).

Inhibition of Plant and Microbial Amidases

Propanil hydrolysis is inhibited by various carbamate and organophosphaleinsecticides in plants (40, 92) and microorganisms (93). Competitive inhibition ofaryl acylamidase activity by these compounds was the basis for increased (synergistic)injury to rice, caused when insecticides were applied to rice in close proximity topropanil application (94). Synergistic effects of propanil with several agrochemicals:carbaryl (1-naphthyl N-methylcarbamate); anilofos IS-[2-[(4-chlorophenyl)( 1-methyl-ethyl)-aminol-2-oxoethyll O,O-dimethyl-phosphoro-dithioate ); pendimethalin [N-( l -€thylpropyl)-3,-dimethyl-2,6-dinitobenzenaminel; and piperophos {S-[2-(2-methyl-l -piperidinyl)-2-oxoethyuO,O-dipropyl phosphorodithioate) in propanil-resistant barn-yardgrass were recently detected using a chlorophyll fluorescence technique (95).Carbaryl's synergism is due to its compeiitive inhibition of aryl acylamidase (40), butthe exact mechanism of the other synergists in propanil-resistant barnyardgrass ispresently unknown.

Interactions of insecticides and soil aryl acylamidase activity have also beenreported (93). When soil was treated with p-chtorophenyl methyl carbamate, propanilhydrolysis was substantially inhibited, and subsequent formation of tetrachloro-diazobenzene was reduced l0- to 100-fold. Carbaryl at t00 pM inhibit€d propanilaryl acylamidase activity by 1O to 70% in several bacte al strains (66, 96\. TheFusarium solani propanil-aryl acylamidases were insensitive to high concenrations ofcarbaryl and parathion (O,Odiethyl-O-4-nitrophenyl phosphorothioare), however thechloroacetanilide, herbicide ramrod (N-isopropyl-2-chloroacetanilide), competitivelyinhibited acetanilide hydrolysis.

74

Carbamate Hydrolysis in Plants

II

Carbamates have a broad spectrum of pesticidal activity and include commonly usedinsecticides, herbicides, nematicides (aldicarb {2-methyl-2-(methylthio)propanal O_[(methylamino)cabonyl]oxime ] and fungicides. There are three major ciasses ofcarbamates: methyl carbamates: aldicarb, carbofuran (2,3_dlhydro-2,2-dimethyl-Z_benzofuranol methylcarbamate), and carbaryl; phenylcarbamates: CIpC, IpC, andphenmedipham; and thiocarbamates: EpTC (S-ethyl dipropyl carbamothioate),butylate [S-ethyl bis(2-merhylpropyl)carbamothioate], and vernoiate (S-propyl diprop_ylcarbamothioate)- The behavior of insecticidal carbamates has been extensivelyreviewed, (97, 98). Studies on IpC (99) and CIpC (100, I1t) in planb indicate thaithe major metabolic route is aryl hydroxylation and conjugation. plants do not cleayethe carbamate bond of the phenylcarbamab herbicides, which is distinct from theinitial metabolism in either microorganisms or animals. The thiocarbamates such asEPTC are metabolized in tolerant species such as corn, cotton (Gossypium hirsutum),and soybeans via initial oxidation to the sulfoxide followed by glutathione conjugation(102, IO3). The pathway for EPTC metabolism in olants is similar to that found inmouse liver microsomes (,104).

Carbamate Hydrolysis in Microorganisms

Microbial degradation of carbamates occurs readily in soil. Accelerated degradationof certain carbamate insecticides has led to ineffectiveness of these compounds, e.g.,control ofphyloxera in vineyards by carbofuran (.105). Bacterial isolates fiom severalgeneta (Art hrobacter, Achromobacter, ATospirillum, Bac i(us, and, pseudomonas) canhydrolyze various insectioidal carbamates (_/06). Hydrolysis is the major pathway forthe initial breakdown of carbofuran, but liule is known about the fate of themetabolites formed by this mechanism (,/06). Mineralization ofthe carbonyl group ofcarbofuran occum more extensively compared to mineralization of the ring struciure(107). The toxicity of aldicarb is greatly reduced when it is hydrolyzed to the oximeand nitrile derivatives (108), but oxidation of aldicarb to the sulfone or sulfoxideyields compounds with similar or greater toxicity. Organophosphorus compounds:paaaoxon (phosphoric acid diethyl-4-nirophenyl ester), chlorfenvinphos [phosphoricacid. 2-chloro- l -(2,-4-dichlorophenyl)ethyl diethyl esterl, and disulfoton (phosphoro_dithioic acid O,O-diethyl S-[2-(etflthio)ethyl]ester] which are esterase inhibitors,suppressed carbofuran hydrolysis in soil for 3 to 2l days (109\. Tlte persistence ofcarbofuran was increased when these esterase inhibitors were combined with thecytochrome P-450 inhibitor, piperonyl butoxide. This synergism is due to rheinhibition of both major mechanisms of carbofuran degradation, i.e., hydrolysis andoxidation.

Some microbial aryl acylamidases fp. striata (l l0), B- sphaericus (59) and acoryneform-like isolate (60)l can hydrolyze certain phenylcarbamates, e.g., CIpC andIPC, as summarized in Table II. However, the p. striata aryl acylamidase is unable tohydrolyze methylcarbanates such as carbaryl. Carbamate hydrolases such as the

75Anhrobacter

. phenmedipham-hydrolase, is specific for phenylcarbamates (1/,/),whereas the Achromobacter carbofuran-hydroiase i. .p""iFo-iJr-_"tnylcarbamates( 1 12\.

.,,^,A jloroli" carbamate_hydrolase has been purified from a pseudomonas sp.(.1,13). This enzym€ is composed of two identical dim"r" *i,f, u ilol""utu. weight of85 kDa each, and is active on carbaryl, carbofuran "na "fai"-i a, ,uUstrates (,flj).The Achromobacter carbofuran-h)droia.. t ̂ U""n purinea oioriogen"ity anO t as "molecular weight of 150 kDa. This enzyme is either cytoplasmic-or occurs in theperiplasmic space, aDd requires Mn* as in actiuator trlil.'rt rrus no urease activityand does not hydrolyze benzamide. The genes ro, it. irnroii*rer carbofuran-hydrolase have been cloned, however they were poorly expressed rr many gram_negative bacteria such as E. coli, Alcalig"ni, "utroph^, i"a i. prAa". This indicatesllu,

*":Iuo processing is required tJ produce ;fil;;"i;;;. The senes forphenmedipham-hydrotase (pcd) ha-ve alio.been cloned unO tf,"'fiot"rn purified tohomogeneity_ (///). The phenmedipham_hydrolase i, u rno"or"i'ii,r, a molecularweight of 55 kDa, and conrains rhe esterase motif fC-f-yii".-X_Cfyl. t"phenmedipham-hydrorase arso has hydrorytic activity o; ,i" -u-iii,ionut

"r,"ru""substrate, p-nitrophenylburyrat€. -fhe pcd gene rv". "*p.""r"J -t

tobacco, andconferred resisrance to phenmedipham'u, .ui.. I O_fofJ fiigi., -than

normat fieldapplication rares (/1J). Arrhough bacteria have been irorut'J- tnui porr"ss divercehydrolytic degradation mechani-sms for cartraryl, few organisms are capable ofcomplete mineralization of the entire molecule. When a b-acterial consortium (twoPseudomonas spp.) was constructed, both hydrolysis ""J-"r"ii"'m**lization ofcarbaryl occuned (/16).Thiocarbamates haye been developed as herbicides (butylate, EpfC andvernolate) and fungicides. Repeated apptiiation of rnese compouiA's to soit led to theuevcropment ol mtcrottal populations with acceleraled rhiocarbamab degradationcapability. Soils adapbd to EPTC degadati"n a.o *piOfy O"gruded a relatedthiocarbamate, vernolate (t lT). However, soils adapted to U"tyfuti aia "ot .ffiy

feerafe E?IC and yernolate U tn. EmC metatoiism o""u.i'in-rnuny genera oftnc.tena (Anhobacter, Baci us, Flavobacteium, pr";ir;;;: ^; Rhod,ococcus )T1., jl3'_1?-"11, um,, paeciromyces, penici ium) l1/ay- iJ- nyorolytic andoxrqauve mechantsms h-ave been proposed for EpTC degradation due to the fact thatqrpropropyfamrne was tound in the media of Arthrob(lcter and Rhodococcus svunsTEI (,//9) and BEI (.f20). Evidence from anothet Rhodocc-"^.ouin fet, inOi"ua""

ll*,ljrl:tl1?:l.of.the nlon1l group of Eprc, foilo;;; ;y-;-dearkylation,rormrng propionaldehyde and N_depropyl_EpTC (121). Cytochrome p-450s,t:.qo"t_'91"- f.o-r _!mc Ndealkylation, havi been ctoned from iii.iit nnoao"o""^strains ( 122-124\.

Organophosphate insecticidesO-dimethyl dithiophosphatel,

Hydrolysis of Organophosphate Insecticides by plants

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

H

76

chromen-7 yl O.O-diethyl phosphorothioate)l have replaced many of the chlorinatedfnsecrfcrdes, because they are more effective and less persistent. Wheat (Triticum:e:tivu:n -L:) :rd sorghvm (Sorghum vulgare L.) rapidly degrade dimethoat€ [O,O_deimethyl-.S-(/V--methylcarbamoylmethyl)phosphorothiolothion'"t"1

to froOu"ts, *t;"tsugg€sts hydrolytic metaborism (,r2i). crude enzyme extract" f.o. *h"u, g".,n .'er"able to hydrclyz€ malathion to. dimethylpho;phorothionare anO Oimettrytptros-phorothiolthioDare (,126). Metabolism of O-etnyi O1+_methyfthioyphenyr j.-propyl_phosphorodirhioare (sulprofos) was studied in "ouon 1f24. ih;;;., merabolireswere the sulfoxide and sulfone deriyatives, indicating o,,idation usi major initialtransformation. Formation of the phenol and gluociside conlugutes of sulprofosindicates hydrolysis_of rhe phospho-phenot bondl b", "";il;;i;;;;;lysis has notbeen confirmed. However, other enzymes, such as mixed-function oxidases andglutathione S-transferases (GST), may Le equally imponant in itre-leto*ittcat;on oforganophosphorus insecticides in plants (.f 2g).

Hydrolysis of Organophosphate Insecticides by Microorganisms

The degradation of organophosphate insecticides has been studied €xtensively inseveral gram-negative bacterial strains, especia y pseudomonas diminuta and inFlavobacterium ATCC 2.1551 (t2g). 'Uydrolysis

of the oiganopf,ospf,orus

ly":f]d:: occurs via nucleophitic addition ofwatei across the acid'innydride bond;tnus tne_ enzymes named parathion_hJdrolases and phosphotriesterases are actually

organophosphorus acid anhydrases (r30). 'the paiathion-nyaroiase can be eithercytosolic or membrane-bound, depending upon the bacterial species. The

Flavobacterium enzymd is membrane-oounal and is a single ""i, .f:j kDa, whereasthe enzyme from strain sc (gram-negative, oxidase-negatiu" u"roui", non-rnotir", ,oosha!:d b^aggliy) is composed of four identical .uU'*io, "u"f, rvi,i a molecularweight of 67 V.0J,^ (129). This enzyme is also a mernbrane-Oo"nJ p-"i" (t2g). Acoumaphos-degrading Nocardia isotate, B_l (/3/) produces a cytoilasmrc parathionhydrolase composed of a single 43 kDa subunir tiZq;. fn"r"'r"iufr, indicate thatenzymes, possessing very diverse characteristics can have the same catalytic function.The aryldiphosphatase gene from Nocardia stain B_l (adp-;;n;t;as nothing incommon with opd genes from other sources, and t"i rno'st likeiy undergoneindependent evolution (t 32).

,. . The bacterial parathion-hydrolase genes (opd) have been cloned from p.

atmtnuta \tJJ, tJ4) and Flavobacterium A-ICC 27551(135). The nucleotidesequence for the Fktvobacterium and p. d.iminuta opd genes are identical (134, 135).The opd genes-were poorly expressed in E. coli, inain" f touoO,ori"rirn hydrolasewas a much larger protein when expressed in E. coli compared to the nativeFlav.obacterium hydrolase- When thi hydrolase was "^p.".JJ in SteptomycesIividins, it was of similar size to that produced in Flavobacteriurn, bua wassynthesized in larger quantities and was secreted extracellularly (,fJ6). production ofan extracellular hydrolase is ideal for remediation pr*"rr"a

'b""uu"" detoxitication

does not require bacterial uptake.

---

77

Nitrile Hydrolysis in Plants

Nitrile groups are essential moieties in the phytotoxicology of the herbicides,bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), cyanazine [ 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 hydrolysis of the nitrile group produces anamide. The amide is subsequently converted to the carboxylic acid, which may bedecarboxylated. This metabolic pathway occun for bromoxynil in wheat (1321 andfor cyanazine in 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 hydroxylated to the respectivecarboxylate by several speries: Fexibacteriun sp. (140) ar.d Klebsiella pneumoniae(l4l)- The K. pneumoniae utilizes bromoxynil as a niaogen source, rather than acarbon soutce, with 3,5-dibromo-4-hydroxybenzoate accumulating as an end-product.Alternatively, an oxidative pathway, mediated by pentachlorophenol-hydroxylase(flavin monooxygenase) from Flavobacterium sp. ATCC 39723 (currentty classifiedas a Sphingomonas), directly liberates cyanide, forming dibromohydroquinone (,f42).Formation of the hydroquinone derivative, rather than the hydroxybenzoatederivative, renders bromoxynil more prone to complete mineralization. Klebsiellabromoxynil-nitrilase genes (brz) have been cloned, sequenced, and the proteinpurified (/43). The bxz genes have been expressed in plants, resulting inbromoxynil-tolerant plants (,f44). Commercial application of this technology iscurrently being used in cotton and potatoes to produce herbicide-resistant crops.

Role of Phosphatases and Sulfatases in Pesticide Degradation

There is limited literature available on the role of phosphatases and sulfatases inpesticide metabolism. The insecticide endosulfan U,2,3,4,7,7 -hexachlorobicyclo

[2.2.1]-2-hepterc-5,6-bisoxymethylene sulfitel is metabolized yia both oxidative andhydrolytic mechanisms in vitro by the white rot fungus, Phenerochaetechrysosporium (145). Under both nutrient-rich and nurientlimiting conditions,endosulfan is metabolized to endosulfan diol. This indicates a different metabolicroute, catalyzed by a sulfatase rather than a lignin peroxidase. Other studies haveshown that endosulfan diol is also formed by hydrolytic cleavage of endosulfan bystatic cultures of the fungus Trichoderma sp. (.146). These observations also suggesta role for sulfatase in fungal metabolism ofendosulfan.

78

Genetic Engineering of Crops for BialaphodGlufosinateResistance

Numerous phytotoxic ..,ubo,i":^T:lu:ed by microorganisms have been isolated,rqenrrrred and resled for rheir porential ̂ i.oi"ii"rl-.O? r"lese. uialaplos ano;ff:31il:lH:ll,

t:il' ffi ,T:

most successrur. cruro,ii"r.' r-".*onium sart orirerbicides (;rit;;r.'fr:l^"t^^l"* been developed as major commercial

ffi '6u:**;tt"*;*$;d;i.;.,#,.',*:il,:f;;1f trmorety (o yietd rhe ry'_acetyt "ornqo::q f"-_prrrotJ"i*i]lt-.uy * acred on bynyororases and/or transferases royield,th" *ti"" piy,ot.ii" ti,f;;_, Fu hermore,metabotism of rhe peptidyl "o.l:lrr3 bi"l"Fh*,-;;;;.;:"iitl'n""" or ransfo.,n"a

fl:lf, l]"i.[11i ff,ll? ] ffi ,l* ";;; ;;;"' ;"'""i..Tli' kansam i nase.. . Bialaphos is a tripeptide

"compr]:g__"f a unique amino acid. L_2_amino_4-lnyoroxy(methyr)phosphinyllbutyric icia rpp.rl ["teiio t*r'.]""ir, moieties. Thecompound was isolated uo, *,:t,=::l i:,",;!i;;;;;;;;rii"jil,",", r,oO, ̂̂ ostreptomyces hygroscopicus (l4g).^,The natural form of pfrl. ,i..i_i.o.* <a_ppff,and rt was the first reported naturally_occurring uri"o Jj "o"*"'ing a phosphinicgroup. Bialaphos was initiallv found to have ,ir" ,",ir"r,grl"r#)i_n, "in"r"o) ^naantibacteriar activity (t47. 4\,f"ir.nl ,. ;;ilfi";"i-,fi,r. cruo,nin"reversed gowth inhibition caused bv i;ur"pr,* r" ar.;; iuoltiii.' cuttures (tln.

i,'#'L::::i'JJ'1;L?,fl;Hf;::^'r'r'"*" re c ? J i:' 6ii i",i"i,v in r .oapossessed.nong pr,yaio*iJi,ni u",t.l9t

nnrtoto*i"ity by Hoechst Ac showed that itsynthesis of ,ft' ;i;#"*o

thts compound was patenled as a herbicide (/5o).formu I ar i on " i,r, "' "",r #' " i,,ii"J",f r : ilf lril,1. :."ffi llrf;fifmrcroorganisms in soil to ppT (/i,f;, which.is also rapiJry J"g.."rd.llr",, r, hatf_tife of4 to 7 days in soirs (/52, /5J). tn,a tesr of 300 bd;;;.i.;i;;i;o'm sorr. a' strains

lj$fjlo"'-f* to the 2_oxo *"!-s :f lT "jl ,;;,"_;;;;iil). crurosinarerrrr., rs a non_setecrive. posremergence_herbi"id" ".;;;;.; i,oll.or ,n or"t "ro.anc vrneyards, in chemical fallou

dou'n heibicide rf ;;;;; ;#;X,srtuahons' as a preharvest desiccant' as a burn-

weedconrror in nun.g"ii" ;ffi i;ff;"y|:Tjlfi l1j:;lloo,]*"* ( /55). 2ni f61

.. . Bialaphos is absorbed through, -ptu"t b;;;;;.;i sJm. t unrro.ation 1orilT'nil fi i:i,'#?rt:" ";:lit I i ",,p;;' ;';. ;;; i.Jd'' i n pr un,,,oo n

metabolizei oy p"p,ii^", in o"n, Y'u'upt'ot does not inhibit GS. but is rapidtyppr-is not a c's'rnir,il;i;;6;j';:T:,il":[jf,:,fr.ii]'d:3. IT.il:ffilsrn transgenic ptants, which have been transformed *tui ;il;;;; L-pm (t60)..rnere rs atso a Iack of degradative-ry{boh.srn .f "-$i l;;;;nlo.rn"a ptunt.,

#T:.JJ:jl. conversion ro the acetyrahd prod";;,; l;il;;",,""ny alrered for

79

o g H _ o H o H - O Ht t i l / | ;? - 'cHrl_cH2 _CH2{H_C-NH_CH_C_fH_CH_COOHl l l loH NH, tr, cH:

Bialaphos

ol l

cltrP{HfCH2_CH_COOHI IOH NH2

phosphinothricin

ot l

cHrP_CH2_CH2_CIr_COOHl roH NACO_CH,

lr'_Acetyl_phosphinothricin

Figure 4. Chemical structures of bialaphos, phosphinothricin and N_acerylatedphosphinothricin.

80

- _ Biotechnological approaches have been utirized in srudies on the biochemistryof?PT in microorganisms and plants. The biosynthetic puii*uy of Uiufupf,o. f,*be€n completely elucidated using various techniques i/6/). Beginning withprecursors containing three carbon atoms, bialaphos ii prju""j in a complex seriesof o_ver a dozen steps (,f61). one step inuoru". "n u""tyicoa-o"p"nJ"nt ,"u"tion tt utmodifies either demethyl_ppT or ppT. The Dcr gene i, ;;p;;ril;;. ."sistance rol3laphos in S. hygroscopicus, and encodes fo, tt-" """tyt t inr?"r".", which convensl];:":l- ::ylllf-

non-phytoroxic merabolire tiozj- -aim.rgr,

this acetylIil*"r"::

rs not^ctassjtied as a hydrolytic enzyme p?/ re, it do€s form an amide bond::,::i". T"-"11e,

be, susceptible to hydrotytic and/or rransferase activity. Aspornreo out prevtously, deacetylases have been s(udied in plants, microorganrsms, aswell.as in mammalian systems, and nitroacetanilid€ auO",.ut", t uu" U""n utilized tofacilitate assaying their activities (/63)_ Such pf"n, o. *i".oiiui"nzymes could acton N-acetyl-PPT to release the phyroroxrc comDound. prrf.

jlT-tit:11o"*In t"nes impaning_ resisrance ro pp[. cto"ins ;f; th.esisranl gene\oar) rfom 5. hygroscopicus (164) and the transformation of ppf_resistant plants hasbeen accomplished (16J). A similar gene Q)at) *on S. ,iriaiciro_ogenes Ti 494,with the same function, was simultaneously isorated and has also been introduced intovarious plant species (160, 166, I6n. presently, more than 20 crop plant qpecieshaye been transformed for resistance to ppf in this manner. Some of theseg.enetically altered plants are resistant to ppT at rates as high as I fg fra-r (ca. l0 times:T^!:-::1 i"T1f

field apptication .rrte) (t64). rhis indicates i high degree ofIncorporallon ot acetyl transferase expression.The previously known anti-fungal activity of bialaphos and glufosinate was

lTj":l-i::::t1,rn .tfree pathogens (Rhizoctoiia sotani,'Scterotiii nomoeocarpa,

::::!:ti:-.yynl!rat!n) in vitro and in vivo on ppT_resistanr rransgenrc

:r::pfng- 9entgrass (Agrostis palustris), an important turfgnss (/6g). Resultsindicated that bialaphos can simultaneously contiol weeds u,ia fungut pathogens inthis transgenic grass. Furthermore, bialaphos tras antibiotic ""ii"i,y'?!ui"r, n "rlrr;Kiihn that causes rice sheath bligha (t6i), and Mognoportu" griilHerben) Barr(r+d) rnat causes nce blast disease. Substantial suppr"ssi,on of sheath blightsymptoms was reported when bialaphos was applied to transgenic plants which had5:"-',lt_:^.:9,:tln,

R. sola.ni pno.r rs herbicide treatment (liojlli*'"i""a ou".g""i"nce pfa s lbrataphos-resistanr (bar) genel had reduced lesions and otner sympromsof_,rice blast disease after bialaphoi triatment (t7t). I; i;;;;d that thesef,::q:?: T.::Tlit*

by bialaphos and ppT, because the microbes lack the abirity:"^:li1,l^i:r:-Tlize

rhe compounds to non-fungitoxic producrs. Thus, r. appearspossrDre to control some serious diseases by using ,ar_transgenic rice cultivars andbialaphos for weed and disease control. Glufosinite tu, ut.JU""n ,u"""ssfully usedto control the weed r€d rice (a conspecific weed ofcultivated rice) in bar_transformedrice ( 172\.

,"^., _tl:]il1::,:lo.Pm.":" :ligl" among commerciat herbicides in that they haveootn potent antrbrotic and herbicidal. propenies. This dual shategy will no doubt beutilized more widely with the increasing availability ofppT_.erisrint c.oo".

81

Summary and Conclusions

Gene.ally, our understanding of microbial hydrolytic enzymes has been greatlyincreased during the past decade, but information on ptant hydrolytic enzymes is notas advanced. Although advances have been made, most of the information onmicrobial hydrolytic enzymes, has not been focused directly on pesticide metatrolism.Many hydrolytic enzymes have been reported to have multiple activities (amidase,esterase, transferase), but most have not been examined for multiplicity, especiallywith regard to the metabolism of pesticides. Also the knowledge atrout the-precislphysiological role of these hydrolytic enzymes is insufficient. Moreover, informationis needed on enzyme mechanisms and regulation of enzyme activity. Many enzymeactive sites or receptor sites recognize only one sterochemical geometry. Thus,understanding enzyme multiplicity, physiological role, mechanism, and regulation,may lead to the development of more specific regulators (e.g., inhibitors, activators),so that more specific and efficacious pesticidal compounds can be developed using abiorational design. The use of techniques such as protein engineering *uy prouid"additional insight on the relationship ofprotein sructure and substrate specificities ofhydrolytic enzymes in plants and microorganisms.

Many industrial synthetic processes produce racemic mixtures, in which onlyone enantiomer is triologically active. Hydrolytic enzymes have high potential valuein the development of bioprocesses for production ofcompounds uslfui to agricultureand other industries. Enzymes have the unique ability to facilitate stereospecifiitransformations and thus, biosynthetic approaches may be more effective in someindustrial syntheses. The cloning of an Arthrobacter eslerase g€ne thatstereospecifically produces (+) r-chrysanthemic acid, utilized in rhe synthesis ofpyrethroid insecticides, is one example demonstrating this biotechnological strategy.This enzyme occurs in low amounts in ahis bacterium, however cloning and over-expression could permit induss-ial-scale preparation. Certain hydrolyric Jn.y*", -.also being considered for remediation of contaminants, e.g., nitrilases for solventssuch as acetonitrile (/73), amidases for acrylamides (174), and atrazine [6-chloro_N_ethyl-N-( I -methylethyl)- 1,3,5-triazine-2,4-diaminel chlorohydrolase, to degradearazine (see chapter by Sadowsky and Wackett in this volume).

As we have discussed, crop engineering for resistance to herbicides, based uponmicrobial hydrolytic enzymes, is a commercial success for bialaohos andphosphinothricin. Future herbicide technologies may utilize other unique microbialhydrolytic enzymes that can be developed for engineering crop resistance to otherherbicides. Other novel pesticides (fungicides, insecticides and herbicides) may alsobe designed as potent inhibitors of hydrolytic enzymes, or that would be activated ordetoxified by specific plant or microbial hydrolytic enzymes.

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ReFinted from ACS Symposium Series 777Pesticide Biotransformation in Plana dd MicroorganisrnsSimil*ities and DivergencesJ. Christopher Hall, Roben E. Hoaglad, and Robert M. Zablotowicz, EditorsPublished 2001 by American Chemical Society