Hydrolytic Dehalogenases Chemistry of the Latter Reactions Remains to Be Explored

FORSCHUNG 116 CHIMIA 47(1993) Nr. 4 (April)

Chimia 47 (/993) 116-/21 from adjacent C-atoms with the formation © Neue Schwei~er;sche Chemische Gesellschaft of an additional bond between the C-at- /SSN 0009-4293 oms). Many bacteria] pure cultures have been shown to catalyze the reductive dehalogenation ofha]oaliphatic compounds, but, so far, only one organism capable of Microbial Dehalogenation anaerobic reductive dehalogenation of ha]oaromatics is availab]e in pure culture of Synthetic Organohalogen [7]. Some reductive dehalogenations have been shown to be catalyzed by coba]t- and Compounds: nickel-porphyrins [8] while others appear to require specific enzymes, and the bio- Hydrolytic Dehalogenases chemistry of the latter reactions remains to be explored. Substitutive dehalogenation reactions are catalyzed by specific enzymes, the Thomas Leisinger* and Regula Bader deha]ogenases, which replace a halogen substituent with a OH group from H~O. Hydrolytic deha]ogenation is commonly Ahstract. Hydro]ytic removal of halogen substitutents is commonly the first step in the observed as the first step in the aerobic degradation of haloa]iphatic compounds by aerobic bacteria, whereas initial deha]o- degradation ofha]ogenated aliphatic com- genation of aryl halides is rare. Hydrolytic deha]ogenations are catalyzed by specific pounds. Initial removal of halogen sub- dehalogenases, a group of enzymes which has been extensively studied in bacteria and stituents funnels haloaliphatics into cen- which does not seem to occur in mammals. Questions pertaining to the origin and tra] metabolism and enables a number of evolution of dehalogenases in soil bacteria have recently become tractable by the bacteria to utilize these halogenated sub- establishment of dehalogenase gene sequences. At the protein level, new deha]ogenas- strates as carbon and energy sources. es are being discovered and known dehalogenases are being analyzed with respect to their mechanisms of catalysis. Finally, microbia] dehalogenases, either as cells of dehalogenative bacteria or as enzyme preparations, have potentia] for applications in 2. Aromatic Dehalogenases environmental biotechnology and biotransformation. The hydrolytic removal of halogen 1. Introduction ygenolytic dehalogenations are often for- substituents from halogenated aromatics tuitous reactions of enzymes with broad would appear the most straightforward Halogenated organic compounds are substrate specificity. They may yield prod- strategy for initiating the degradation of not only important industria] chemicals, ucts which are not further metabolized by these compounds. Degradative pathways but also significant environmental pollut- the deha]ogenative microorganism. This compatible with this mechanism, howev- ants. They figure as the largest group of metabolic pattern, in which the ha]ogenat- er, have rarely been described, and this is compounds on the U.S. Environmental ed compounds are not utilized as carbon thought to reflect the resistance of haloar- Protection Agency's listofpriority Pollut- and energy sources, is termed cometabo- omatics to nucleophi]ic displacement re- ants [I]. Their wide distribution in the ]ism. actions underphysio]ogical conditions [9]. environment and their often toxic or carci- Reductive deha]ogenation occurs pri- An exception appears to be realized in a nogenic potentia] have prompted exten- mari]y in anaerobic environments. For number of bacterial isolates representing sive studies on their degradation by micro- several highly chlorinated industrial com- the genera Pseudomonas, Arthrobacter, organisms, the principal agents for the pounds, including polychlorinated biphe- and Corynebacterium which were shown ultimate breakdown of organic compounds nyls, pentachlorophenol, and tetrachlo- to initiate the degradation of 4-chloroben- in nature. Microbia] organohalogen me- roethene, it provides the only known bio- zaate by displacement of C1 through OH. tabo]ism and its potential and limitations degradation mechanism. Reductive dehal- The fact that the enzymatic conversion of for applications in environmental biotech- ogenation results either from hydrogenol- 4-chlorobenzoate to 4-hydroxybenzoate nology have been summarized and dis- ysis (rep]acement of a halogen substituent occurred in the absence of molecular 0, cussed in excellent reviews [2-7]. with an H-atom) or from diha]oelimina- [10-12], and incorporation experiments 18 18 Microorganisms use three principal tion (removal of two halogen substituents with 02 andH2 0 [13][14] demonstrat- mechanisms for C-halogen bond break- age: oxygenation, reduction, and substitu- Scheme I. Dechlorination of4-Chlorobenzoate by Pseudomonas sp. CBS3 (from [17][ 18]). Reactions tion. Oxygenative reactions figure most 1,2, and 3 are catalyzed by 4-chlorobenzoate: CoA ligase, 4-chlorobenzoyl-CoA dehalogenase and prominently in the degradation of haloar- 4-hydroxybenzoyl-CoA thioesterase, respectively. The system is currently registered as EC 3.8.1.6. omatics. Many oxygenases non-specifi- cally oxygenate haloaromatics and gener- ate unstable intermediates from which COOH CO-SCoA CO·SCoA COOH halogen is spontaneously eliminated. Ox- ATP,Co.ASH. "2° "20 "\ • ~ '" ¢ "AUP, PPj ¢ ""'-.-"e 1 ¢ CoASH ¢ *Correspondence: Prof. Dr. T. Leisinger CI CI OH OH Institute of Microbiology ETH-Zentrum 2 3 CH-8092 Zurich, Switzerland FORSCHUNG 117 CHIMIA ~7( Iqq.l) Nr. 41April) ed that the OH group displacing CI origi- Table 1. Aliphatic Dehalogenases nated from H20 and not from molecular H20 02' The hydrolytic dehalogenation of 4- Haloalkanoare Dehalogcnase: R ·CHX 'COOH~ R-CHOH -COOH chlorobenzoate was studied in detail with HX Pseudomonassp. CBS3. The4-chlorobenzoate dehalogenase of this organism turned out to be a substrate-inducible multi-com- H20 ponent system accepting 4-chloro-, 4-bro- Haloalkane Dehalogenase: R - CH2 - CH2X ~ R - CH2 - CH20H (R = H; CH3; [CH2]n - CH3' n=I.13) 010-, and 4-iodobenzoate but not 4-fluor- HX obenzoate as substrates. The genes encoding the dehalogenase proteins have been cloned and expressed in E. coli [15][16]. GSH H20 They encode a 57-, a 30- and a 16-kD Dihalomethane Dehalogenase: CH2X2 ~ [GSCH2X ~ GSCH20H] T HCOH polypeptide [16]. Purification of these pro- HX HX GSH teins from recombinant E. coli led to the assignement of 4-chlorobenzoate CoA ligase, 4-chlorobenzoyl-CoA dehaloge- Hx ~°, nase, and 4-hydrox ybenzoy I-CoA thioeste- Haloalcohol Dehalogenase: R • CH2 - CHOH - CH2X ~ R - CH2 - CH - CH2 (R = H; X) rase to one each of the three polypeptides HX and to the proposal of the 4-chlorobenzoate degradation pathway shown in Scheme 1 [17]. The properties of the re- H20 cently purified 4-chlorobenzoate CoA 3-Chloroacrylic Acid Dehalogenase: HOOC - CII = CHX ~ HOO<:. CH2 . COH ligase from cell extract of Pseudomonas HX sp. CBS3 [18] SUppOlt this sequence of reactions. Dehalogenation of 4-chlorobenzoate Table 2. Haloalkanoic Acid Dehalogenases thus is based on an ATP-consuming reac- Enlyme • ub. trale ub~.tralclPrl dUCI tion. Adenylylation of the carboxyl group nfigurali n of 4-chlorobenzoate, followed by displacement of the AMP with a thiol group from HaloucellllC Dehalogenase coenzyme A leads to the formation of a group ,\ I ch loroucclulC thioester. This activated intermediate fa- nu roucelalc cilitates nucleophilic attack by OH- from group \2 hloroucel:lle HzO at C(4) of the aromatic ring and leads to the displacement of the halogen substit- _-Huloacid chalQgcna e uent. Degradation of aromatic acids via group 81 L-2-halouclds inVl.'rsion the formation of coenzyme A esters is well gr up B_ D- and I -2-haloacid~ in\" 'r.lon known in anaerobic bacteria [19]. Its dis- group 83 lJ- and L-_-hulollCld~ retcnti n tri bution among aerobic, 4-chlorobenzoate group 8.J o-2-haloucld invcrsion utilizing bacteria remains to be established. There is circumstantial evidence for its occurrence in at least two other bacteria utilizing a haloaliphatic compound as a haloacetates only and the 2-haloacid de- [II ][20]. On the other hand, labelling ex- growth substrate are obtained, is correlat- halogenases (EC 3.8.1.2.) which act on 18 peri ments wi th H2 0 suggest that hydro- ed with the occurrence of this substrate in haloacetates, 2-halopropionates and in lytic dehalogenation of 4-chlorobenzoate the environment, but there are no system- some cases also on 2-haloalkanoic acids in cell extracts of an Arthrobacter sp. does atic studies on this aspect. In general, and of higher C-chain length. As shown in not proceed via a thioester [14]. where this has been examined, aliphatic Table 2, each of these groups is further dehalogenases react at increasing rates divided into subgroups [5]. In the case of with the 1-, Br-, and CI-substituted analogs the haloacetate dehalogenases, the classi- 3. Aliphatic Dehalogenases of a particular compound. Fluorinated com- fication is based on the reactivity of the pounds, however, are usual Iy not attacked. enzymes towards 2-fIuoroacetate. Among The reactions catalyzed by aliphatic These observations are in accordance with the 2-haloacid dehalogenases, four sub- dehalogenases are shown in Table 1. Rep- the stability of the C-halogen bond which groups can be recognized on the basis of resentatives of the enzymes listed have increases in the order 1< Br < CI < F. The their stereospecific action on D,L-2-mon- been purified to homogeneity and charac- enzymology, biochemistry, and genetics chloropropionate and the stereochemical terized to various extents. Gram-positive of aliphatic dehalogenases have recently configuration of the product [22]. As evi- or Gram-negative soil bacteria capable to been reviewed by Hardman [5]. dent from Table 3, the subunit molecular grow on a particular haloaliphatic com- mass ofhaloalkanoate dehalogenases rang- pound served as enzyme sources. In some 3.1. Haloalkanoate Dehalogenases es between 25 and 34 kD. Depending on cases such organisms were readily isolat- Haloalkanoate dehalogenases are the the enzyme, monomeric, dimeric, and te- ed from ordinary soil, whereas in other most widely studied hydrolytic dehaloge- trameric tertiary structures have been re- cases isolates were obtained only from nases. Their discovery dates back some 30 ported. enrichment cultures inoculated with sam- years [21]. There are two major groups of The details of the dehalogenation reac- ples from polluted environments. One as- these enzymes: the haloacetate dehaloge- tions catalyzed by 2-haloacid dehaloge- sumes that the ease with which organisms nases (EC 3.8.1.3) which are active on nases have not been elucidated, but possi- FORSCHUNG 118 CHIMIA 47 (1993) Nr. 4 (April)

ble mechanisms have been proposed. Two of multiple dehalogenases is currently second and the third class exhibited se- different mechanisms are considered for being explored by studying the expression quence homology with haloalkane or enzymes that lead to inversion of configu- of dehalogenase genes at the molecular dichloromethane dehalogenase or with ration during the reaction (Table 2, groups level and by assessing their evolutionary other proteins whose sequences are stored B1, B2 and B4). One model proposes that relatedness. The majority of the haloal- in databases [23][26-28][30]. It is inter- an enzyme active site with an electron- kanoate dehalogenases is substrate-induc- esting to note that in two of the three cases, donating group leads to the activation of ible, but several cases of constitutive en- where an organism contains two haloal- water which directly displaces the halo- zyme formation have also been reported. kanoate dehalogenases, these enzymes gen in a nucleophilic substitution reac- Little is known about the regulatory mech- belong to different evolutionary classes. tion. In the other model, a carboxylate anisms involved in dehalogenase expres- Nevertheless the corresponding genes are group from an aspartate or a glutamate at sion. In one instance, preliminary evi- arranged adjacently [27] or in close prox- the active site acts as the nucleophile. This dence suggests negati ve control by a re- imity [25] to each other. would lead to the formation of an ester pressor [26], in an other there are indica- intermediate which is hydrolyzed by the tions for positive control by an rpoN- 3.2. Haloalkane Dehalogenases attack of water on the carbonyl C-atom dependent activator and for transcription Haloalkane dehalogenases (EC 38.1.5) [5][22][23]. Enzymes whose activity re- from a -24/-12-type promoter [29]. The form another important group of halido- sults in retention of configuration (Table recent esthablishment of eight nucleotide hydrolases that so far has exclusively been 2, group B3) are highly sensitive to sulfhy- sequences encoding haloalkanoate dehal- observed in bacteria. These enzymes hy- dryl-blocking agents. This observation has ogenases provides information on the ev- drolytically convert halogenated aliphatic led to a proposal involving a sulfhydryl olutionary relatedness of these enzymes. hydrocarbons and related compounds to group in the active site, displacement of Table 4 shows the amino acid identities of the corresponding alcohols (Table 1). Or- halogen by formation of a thioether-en- eight haloalkanoic acid dehalogenases ob- ganisms producing haloalkane dehaloge- zyme intermediate and retention of conserved in pairwise alignments of the de- nases have been obtained from soil by figuration through a double inversion ducedarnino-acid sequences. The enzymes enrichment cultures using 1,2-dichloro- [22][24]. Comparison of the conserved fall clearly into three classes which exhibit ethane, l-chlorobutane, l-chlorohexane, regions in the haloalkanoate dehaloge- low (12-21 %) identity to each other and, or 1,6-dichlorohexane as the sole source nase gene sequences available so far (Ta- thus, have probably evolved form differ- of carbon and energy. The isolation and ble 3) do not reveal the details of the ent ancestral proteins. One class is repre- the characterization of halo alkane dehalo- dehalogenation mechanisms realized in sented by the group A 1 haloacetate dehal- genases have been reported from the Gram- the different systems. The gene sequences ogenase of Moraxella sp., strain B. This negative X. autotrophicus GJlO [32] and do, however, open possibilities for ap- enzyme shares two conserved regions in from the gram-positive bacteria Rhodo- proaching this question by in vitro site- the N-terminal part with three hydrolases coccus sp. HAl [33] (reassigned from directed mutagenesis. of P. putida and with the haloalkane de- Arthrobacter after chemotaxonomy by Bacteria utilizing haloalkanoic acids halogenase of Xanthobacter autotrophi- DSM), Corynebacterium sp. strain m15-3 as growth substrates often contain two or cus [25]. The second class comprises six [34], strain Gl70 [35] and Rhodococcus even three [31] haloalkanoic acid dehalo- enzymes, namely the one group A2 and the erythropolis Y2 [36]. Common properties genases, specific for either a single stere- five group B1 dehalogenases that have of these enzymes include their monomeric oisomer of the halogenated substrate or been sequenced so far. Percent identity structure and their relative molecular mass active against both isomers (Table 3). It within this class ranged from 33-67%, which ranges between 28 and 36 kD. With has been suggested [5] that possession of and the sequences shared amino acid iden- the exception of the X. autotrophicus en- more than one dehalogenase gene confers tities at 46 positions. The group B4 dehal- zyme, the haloalkane dehalogenases were flexibility under fluctuating environmen- ogenase of P. putida All represents the found to be inducible. The preferred sub- tal conditions and thereby selective ad- third distinct class of dehalogenases. None strates of these enzymes as a group are vantages on an organism. The significance of the haloalkanoate dehalogenases in the mono- and biterminally halogenated al-

Table 3. Haloalkanaic Acid Dehalogenases and Their Genes in Soil Bacteria

~hal gtlnas Gene L ali n r gen D.!duc: d prO! in (grouP)') (de jnnmion) molecular rna .b) (I.,D)

\'f(/((l\t!lIu "p.. train B I deltHI pia mId 33. 25 A2 dehHl plasmiu _6.0

PWI/(/wl/(ma.,p B' 81 ridl chrolm om~ 25.4- 81 d/!/tCII eh!' rn lOll' , .6

PII IIclrllll/11II11 fill/ida 81 /melL 'hrlllll0S0mc 1<;,7 27 M hadD 'hrom <; me •.. 6 2

83 de/rl trnn\[1u~()n 29 82 ele/III tmn'po~on 29

P'I'lIdII/lWlIlI' ct'prtda 1B of 8/ hcffl II chromos me _5.9 30

, l/1/1hoh lcta rlIl1r1lrt>flhkll.\ J lOB / dhiB chrom ~ me

") (f Table; b) Molecular mass is given only for those enzymes whose structural genes have been sequenced. FORSCHUNG 119 C'HIMIA 47 (1993\ Nr. 4 (Aprill

Table 4. Percent Identitya) between the Deduced Amino-Acid Sequences of Bacterial Haloalkanoate Dehalogenases

l:n/ymcs Genc~) ImdD dhiB I1dfrvn hadL dehen d/!lrCl dchm delrlll gmup<)

I deh"l 16 15 19 16 16 12 19 100

l2 dehH2 17 44 33 49 4-9 .5 100

81 dehe! 4-2 67 3 36 (00

BI dl'hell 11 42 37 SO 100

HI hadL ]9 43 36 100

BI htlll a 21 40 100

81 dlrlB 14 100

B-1 l/(/dD 100 a) Percent identity was determined by the program GAP (Genetics Computer Group, University of Wisconsin, Madison). b) For references of the nucleotide sequences, see Table 3. C) Cf Table 2. kanes of chain lengths up to 16 C-atoms. nucleophilic residue essential for cataly- responsible for inducibility of the dehalo- Unsaturated or branched haloalkanes are sis. This system, thus, has been developed genase by dichloromethane. dcmR encodes also accepted, as are secondary haloal- to a point where further work should lead a trans-acting factor which negatively con- kanes, haloalcohols, and the environmen- to a detailed understanding of the en- trols dichloromethane dehalogenase for- tally relevant compounds MeBr, 1,2- zyme's reaction mechanism as well as to mation at the transcriptional level [42]. dibromoethane, 1,2-dichloroethane, and the engineering of enzyme variants with Analysis of the amino acid sequence de- bis(2-chloroethyl) ether [5][35]. None of altered substrate specificity. duced from dcmA showed that dichlo- the enzymes dehalogenates alkanes with romethane dehalogenase belongs to the more than two C-atoms carrying vicinal 3.3. Dichloromethane Dehalogenase glutathione S-transferase (GST, EC CI substituents, and none of them exhibits Dichloromethane dehalogenase is a 2.5.1.18) enzyme family [4l].The same chiral specificity. strongly inducible enzyme enabling fac- holds true for the dichloromethane dehal- According to their substrate range the ultatively methylotrophic bacteria to grow ogenase of the restricted facultative bacterial haloalkane dehalogenases char- on CH2Cl2 as the sole carbon and energy methylotroph Methylophilus sp. DMI I acterized so far appear to fall in two class- source. It transforms CH2Cl2 to inorganic whose structural gene we have recently es. One class is formed by the X. au- chloride and formaldehyde, a central me- sequenced (R. Bader, unpublished). totrophicus enzyme which is active to- tabolite of methylotrophic growth. In con- Soluble GSTs are a supergene family ward C1-C4 haloalkanes, particularly to- trast to the haloalkane dehalogenases, nu- of proteins which catalyze the conjugation ward the important pollutant 1,2-dichlo- cleophilic displacement of CI by dichlo- of GSH to a variety of electrophiles, and roethane. The other class encompasses the romethane deha]ogenase is not based on these enzymes have been extensi vely stud- enzymes of the Gram-positive organisms the direct attack by OH- or by a carboxy- ied in eukaryotes [43]. Based on amino- which dehalogenate ] ,2-dichloroethane at late group at the enzyme active site, buton acid sequence similarity, eukaryotic GSTs a negligible rate, but are active toward the thiol group of the tripeptide glutath- have been placed into four classes, namely long chain haloalkanes. Similarity in the ione (GSH) (Table 1). Dichloromethane Alpha, Mu, Pi, and Theta [44]. GSTs have N-terminal amino acid sequences (at least dehalogenase was purified and character- also been purified and characterized from ]7 of the first 18 amino-acid residues are ized from five dichloromethane utilizing bacteria, but dichloromethane dehaloge- identical) has been observed between the methylotrophs [39][40]. The dehalogenas- nase is the only prokaryotic GST for which enzymes from Rhodococcus sp. HAl and es were strictly GSH-dependent, hex am- an association with the GST family has R. erythropolis Y2 [33][36]. This may eric enzymes with a subunit molecular been shown at the functional as well as at indicate evolutionary relatedness of these mass of between 33 and 35 kD. They the structural level. Sequence compari- two enzymes and the possible distribution exhibited a narrow substrate range, react- sons indicate a particularly close relation of a haloalkane dehalogenase structural ing with dihalomethanes only, and their of the two bacterial dichloromethane de- gene among Gram-positive bacteria by relatively low catalytic activity was com- halogenases with the Theta class of eu- horizontal transfer. The haloalkane dehal- pensated for by their high intracellular karyotic GSTs. This observation has giv- ogenase from X. autotrophicus has been concentrations (up to 20% of tot a] soluble en rise to speculations [44] on the evolu- studied at the molecular level. Its structur- protein). tion of GSTs and on a possible role of al gene has been sequenced [37], the pro- The genes for dichloromethane utili- bacterial dichloromethane dehalogenase tein has been crystallized, and its three- zation were studied in Methylobacterium as the archetypal enzyme of the GST en- dimensional structure has been determined sp. DM4. They are encoded by a 2.8 kb zyme family. at 2.4-A. resolution [38]. The structural chromosomal DNA fragment whose nu- data suggest that none of the four cysteine cleotide sequence has been determined 3.4. Haloalcohol Dehalogenases residues in the polypeptide chain is in- [4]]. This DNA region containsdcmA, the Haloalcohol dehalogenases or halohy- volved in catalysis. Rather an aspartate at structural gene of dichloromethane dehal- drin hydrogen-halide lyases have been the putative active site seems to act as the ogenase, and dcmR, the regulatory gene purified and characterized from Arthro- FORSCHUNG 120 C'HIMIA 47( 1993) Nr. 4 (April)

Scheme 2. Bacterial Degradation of Halohydrins and Epichlorohydrin. Steps] to 5 are catalyzed by 3.5. 3-Chloroacrylic Acid the following enzymes or bacterial cultures: 1) Haloalcohol dehalogenase from Arthrobacter sp. AD2 Dehalogenases [45], haloalcohol dehalogenase from Corynebacterium sp. N- 1074 [46][48].2) Utilization of (R)-2,3- 3-Chloroacrylic acid dehalogenases dichioropropan-I-ol by Pseudomonas sp. DS-K-29 [15]. Utilization of (S)-2,3-dichloropropan-l-ol have been detected in two unidentified by Alcaligenes sp. DS-K-S38 [52]. 3) Epoxide hydrolase in crude extracts of Corynebacterium sp. N- 1074 [48] and Pseudomonas sp. AD] [53].4) Haloalcohol dehalogenase from Arthrobacter sp. AD2 coryneform bacteria [49][50] and in aPseu- [45], utilization of (R)-3-chloropropane-I,2-diol by Alcaligenes sp. DS-S-7G [54], utilization of (S)- domonas cepacia strain [49]. These or- 3-chloropropanedi-1 ,2-01 by Pseudomons sp. DS-K-2DI [55]. 5) Epoxide hydrolase in crude extracts ganisms were obtained by enrichment on of Pseudomonas sp. AD] [53]. media containing 3-chloroacrylic acid as the sole carbon source. The hydrolytic C1CH2- CHOH - CH2C1 C1CH2- CHCI - CH20H dehalogenases discussed so far do not 1,3-Dichloro-2-propanol 2,3-Dichloro-1-propanol accept substrates possessing halogen substituents on unsaturated C-atoms. Induci- ble enzymes exhibiting this type of activity, however, were detected in crude extracts of the 3-chloroacrylic acid utilizing bacteria. Each of the two coryneform bacteria produced two dehalogenases, one specific for cis- and the other for trans-3- "o, chloroacrylic acid. The P. cepacia strain C1CH2- CH - CH2 was capable of growth exclusively with the cis-isomer and accordingly contained Epichlorohydrin a cis-specific enzyme only. The enzymes form malonate semialdehyde (3-oxopro- 3 ["'" pionic acid) from their respective substrate (Table 1). The dehalogenation of 3- chloroacrylic acid is thought to proceed by C1CH2- CHOH - CH20H hydration of the C=C bond, yielding the 3-chloro-1,2-propanediol unstable intermediate 3-chloro-3-hydroxy- Hel propanoic acid from which HCI is eliminated to give malonate semi aldehyde 4 t [49][50]. o / "- The enzymes of the coryneform bacte- CH20H - CH - CH2 rium FG41 were purified and character- Glycidol ized. cis-3-Chloroacrylic acid dehalogenase was found to consist of two 16.2 kD polypeptide chains whereas trans-3-chlo- 5 ["'" roacrylic acid dehalogenase was composed of7.4 and 8.7 kD subunits whose arrange- CH20H - CHOH - CH20H ment in the native enzyme is not known. Each enzyme was completely specific for Glycerol its respective isomer of 3-chloroacrylic acid, and no other substrates have been found so far [49]. bacter sp. strain AD [45] and from Co- Corynebacterium have revealed a hitherto rynebacterium sp. N-1074 (46). These in- undetected catalytic reaction of this pro- 4. Applications ducible enzymes catalyze the dehalogen- tein, namely its ability to transform, in the ation of vicinal mono- and dichlorinated presence of cyanide, epoxides into /3-hy- The application of bacteria possessing alcohols to their corresponding epoxides droxynitriles [47]. Neither of these en- hydrolytic dehalogenases comprises two (Table 1). Substrates accepted by the enzymes exhibited enantioselectivity. How- major fields: Treatment processes for in- zymes included, in the order of decreasing ever, more recently a second haloalcohol dustrial waste streams and biotransforma- activity, ] ,3-dich10ropropan-2-01, 2-bro- dehalogenase has been purified from Co- tions for the production of chiral building moethanol, l-chloropropan-2-01, 3-ch10- rynebacterium sp. N-1074. This enzyme blocks. ropropane-] ,2-diol, and chloroacetone. No is a heterotetramer composed of 32- and Biotechnological processes have been dehalogenase activity was detected with 35-kD polypeptides. It showed some enan- developed to remove dichloromethane epichlorohydrin (3-chloro-] ,2-epoxypro- tioselectivity and converted 1,3-dichloro- from industrial effluents and waste gases. pane) or 2,3-dichloropropan-l-ol. Both propan-2-01 to (R)-epichlorohydrin of7 5% In aerobic fluidized bed reactors inoculat- enzymes catalyzed the reverse reaction of optical purity [48]. Like many of the halo- ed with dichloromethane utilizingmethylo- dehalogenation, i.e. the halogenation of alkanoate degrading bacteria (Table 3) trophic bacteria and fed with model [56] or epoxides to the corresponding haloalco- Corynebacterium sp., thus, also contains process wastewater [57], this compound hols. The Arthrobacter and the Coryne- two dehalogenases catalyzing the same was mineralized at rates of up to 1.6 I bacterium enzymes have a subunit molec- reaction but differing from each other with g/I/h- • Waste gases containing CH2Cl2 ular mass of 29 and 28 kD, respectively. respect to enantioselectivity, molecular have been treated successfully in aerobic The former is reported to be.a dimer [45], mass, substrate specificity and immuno- trickling-bed reactors and biofi lters while the latter appears to be a tetra mer logical properties [48]. [58][59]. 1,2-Dichloroethane is the other [461. Studies with the pure enzyme from important environmental chemical whose FORSCHUNG 121 CHIMIA ~71IQQ.\) Nr. 41Aprill

bacterial degradation in bioreactors has [I] T. Leisinger, Experientia 1983, 39, 1] 83. [31] lA. Leigh, lA. Skinner, R.A. Cooper, been studied [60][6]]. As in the case of [2] W. Reineke, HJ. Knackmuss, Ann. Rev. FEMS Microbiol. Lett. 1988, 49. 353. [32] S. Keuning, D.B. Janssen, B. Witholt . .I. CH2CI2, the results obtained in pilot stud- Microbio/. 1988,42,263. ies were encouraging, and these systems [3] T.M. Vogel, C.S. Criddle, P.M. McCarty, Bacterio/. 1985,163,635. show promise for large scale application Environ. Sci. Techno/. 1987,21, 722. [33] R. Scholtz, T. Leisinger, F. Suter, A.M. Cook, J. Bacteriof. 1987, 169,5016. in the cost-effective treatment of contam- [4] L.P. Wackett, in 'Applications of Enzyme Biotechnology', Eds. J.W. Kelly and T.O. [34] T. Yokota, T. Omori, T. Kodama. J. Bacte- inated groundwater and process water. Baldwin, Plenum Press, New York, 1991, rial. 1987, 169,4049. Microbial dehalogenation reactions p. 191. [35] D.B. Janssen, J. Gerritse. J. 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