potential pharmaceutical compounds from dioxygenase...

389Current Trends in Biotechnology and PharmacyVol. 6 (4) 389-397 October 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)

Potential Pharmaceutical Compounds fromDioxygenase-Derived Chiral Metabolites

Hamdy A. HassanEnvironmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute,

Menoufia University - Sadat city - Egypt*For Correspondence - [email protected]

Dioxygenase-Derived Chiral Metabolites

Abstract Aromatic and chlorinated aromatic

compounds are used in large quantities asherbicides, pesticides, and solvents, anddistributed in the biosphere due to these industrialactivities contamination. A broad range ofperipheral reactions results in a restricted rangeof central intermediates, which are subject to ring-cleavage and funneling into the Krebs cycle. Keyenzymes in aerobic aromatic degradation areoxygenases, preparing aromatics for ring-cleavage by the introduction of hydroxyl functionsand catalyzing cleavage of the aromatic ring. Theregio- and stereo-specificity of dioxygenaseenzymes revealed that these enzymes are animportant class of biotechnology. These Enzymesthat are capable of catalyzing the insertion ofoxygen into aromatic substrates have manypotential applications in pharmaceuticalsmanufacturing, production of chemicals and alsoin medicine. The regio- and stereospecificoxidation of an unactivated aromatic compoundis very difficult to accomplish using conventionalchemical techniques, which typically produce anarray of byproducts that must be separated anddestroyed. Their potential for derivatizationthrough arene functionalities makes cis-dihydrodiols valuable synthetic building blocks forthe synthesis of biologically important pinitols,conduritols, and acyclic as well as the drugsindinavir and pancratistain, screening methodsfor Dioxygenase enzymes, a product of anoxidation reaction is converted into a phenol or a

catechol, which is easily detected by a Gibbsassay. This conversion allows for a sensitive andefficient assay. Also methods for detectingphenolic ether-products and sulfhydryl productsfrom oxidation reactions by using a Gibbs assay..Keywords: Ring hydroxylating dioxygenase, cis-dihydrodiol, Indinavir, Indigo.

Introduction

Most of building blocks of biomass arearomatic compounds, which are widelydistributed from low-molecular mass compoundsto polymers in nature, and mostly are found asaromatic amino acids, xenobiotic compoundsand lignin components in higher plants, which issecond most abundant polymer in nature aftercellulose, comprising about 25% of the land-based biomass on Earth (1). The recalcitrantorganic matter formations in soils is due to lignindegradation, aromatic compounds from otherplants and decomposition process. The aromaticcompounds are the most stable and persistentorganic pollutants which comes from aromaticamino acids and extensive use of natural andxenobiotic aromatic compounds in industrialprocesses, in addation to inadequate wastemanagement strategies.

The degradation of aromatic polymers isan important component of globalbiogeochemical cycles and is accomplishedalmost exclusively by microorganisms which play


important roles in the degradation andmineralization of xenobiotic and aromaticcompounds in natural environments and suchcapabilities can be used for the clean up ofcontaminated environments (bioremediation).Bioremediation is considered as a relatively low-cost technology, which usually has a high publicacceptance and can often be carried out on site.Bacteria have evolved diverse strategies todegrade aromatic compounds using its hugecatabolic diversity relaxed substrate specificityof some of the catabolic pathways, and therebyderive carbon and energetic benefit from them.Two key steps for the bacterial degradation ofhydrophobic aromatic pollutants is usuallyinitiated by dioxygenases, which utilize molecularoxygen as a required substrate adding bothatoms of O

2 to the aromatic ring. In general, this

reaction is the most difficult in the degradation ofaromatic compounds, and the addition ofhydroxyl groups to the highly stable aromatic ringstructure activates the molecule for the furthersecond step which is oxidation and eventual ringcleavage. The activation of aromatics is usuallycatalyzed by members of the super family ofRieske non-heme iron oxygenases.

Rieske non-heme iron oxygenases : Membersof this super family are known to overall oxidizehundreds of substrates including linked and fused

aromatic, aliphatic olefins, and chlorinatedcompounds and are distributed among a varietyof Gram-negative and Gram-positive bacteriacapable of degrading key classes of aromaticpollutants. Rieske non-heme iron oxygenasesare soluble, multicomponent enzyme systemscomprising two or three separate proteins, andrequire oxygen, ferrous iron (Fe2+) and reducedpyridine for catalysis. These enzymes consistsof an electron transport chain (Fig. 1), thatchannels the electrons from NAD(P)H to thecatalytic terminal oxygenase component wheresubstrate transformation take place (2, 3, 4).These terminal oxygenase component anddifferent electron transport proteins, usuallycatalyze the incorporation of two oxygen atomsinto the aromatic ring to form arene-cis-dihydrodiols (Fig. 2) a reaction which is followedby a dehydrogenation catalyzed by cis-dihydrodioldehydrogenases to give (substituted) catechols.Since decades, Members of the Rieske non-heme iron oxygenases are known to be involvedin benzoate degredation (5), then 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene (benzoate-cis-dihydrodiol) was formed (6). Similar two-component enzyme systems (Fig. 1) areresponsible for 1,2-dioxygenation of anthranilate(7), an intermediary metabolite of tryptophandegradation and a precursor for the

Fig. 1. The bacterial degradation of hydrophobic aromatic pollutants is usually initiated bydioxygenases, which utilize molecular oxygen as a required substrate adding both atoms of O2 to thearomatic ring to form cis-dihydrodiol.



Hamdy A. Hassan

Pseudomonas quinolone signal (8). Many Rieskenon-heme iron oxygenases have beencharacterized, and classified, its classificationsystem was based on the components of theelectron transfer chains present in the Rieskenon-heme iron oxygenase systems either two orthree, this classification system was rathersuitable as long as only a small number ofenzymes were known. Two-component(reductase, oxygenase) and three-component(reductase, ferredoxin, oxygenase) enzymesystems could be differentiated and theseclasses were further subdivided based on thenumber of proteins comprising the oxygenase,the type of flavin moiety (FAD or FMN) presentin the reductase, the presence or absence of aniron-sulfur center in the reductase, and the typeof iron-sulfur center present in the ferredoxin.However, with the increasing number anddiversity of enzyme systems characterized andthe presence of new enzymes with unusual redox

partners that do not fit into the originalclassification it became obvious that suchclassification was not useful anymore. Moreover,it became clear, that the components of theelectron chain are not determining substratespecificity, but was to a certain extentinterchangeable between different Rieske non-heme iron oxygenase systems. Werlen et al (9)proposed a classification system based onsequence alignments of the oxygenase ásubunits, differentiating four families(naphthalene, toluene/benzene, biphenyl, andbenzoate/toluate). Since the oxygenase is thecatalytic component and the subunit plays amajor role in determining substrate specificity (10,11) these classifications are based on thecatalytic activity of the enzymes. Further analysisbased on -subunit sequence comparisonsconfirmed that the grouping of the oxygenaseslargely correlates with the respective substratepreferences (2, 12). Also Gibson and Parales

Fig. 2. The potential of cis-dihydrodiols for derivatization through arene functionalities makes them valuable synthetic building blocks for chiral drugs and specialty chemicals.


distinguished four families. Group I, or thephthalate family, comprises Rieske non-hemeiron oxygenases that contain only subunits.Substrates for this diverse group of enzymesinclude several aromatic acids such as phthalate,p-toluate, and phenoxybenzoate, but alsocarbazole and 2-oxo-1,2-dihydroquinoline. GroupII, or the benzoate family represents a cluster ofenzymes with activities toward various aromaticacids. Naphthalene, phenanthrene, andnitroarene dioxygenases clustered as group IIIand were termed naphthalene family. Biphenyl,toluene and benzene dioxygenases wereobserved to be highly similar in sequence andthus grouped as one cluster (Group IV or thetoluene/biphenyl family). With the increasinginterest in microorganisms capable to degradearomatic pollutants as well as naturally occurringaromatics, however, various enzymes werecharacterized in the recent years, which were onlydistantly related to above described oxygenases(4). Some initial dioxygenases and other catabolicgenes share the chain for the transport ofelectrons as in case of the naphthalenedioxygenase and and salicylate 5-hydroxylase(13).

Pharmaceutical applications of dioxygenase:Aromatic-ring-hydroxylating dioxygenases haveproven useful in a number of biotechnologyapplications. The basis for most applicationsdepend on the stereoselective cis-dihydroxylationof nonactivated aromatic compounds, which isunique to this enzyme while they catalyze animpressive array of different reaction types.Examples include dioxygenase-catalyzedsynthesis of chiral intermediates for thepreparation of natural products, polyfunc-tionalized metabolites, and pharmaceuticalintermediates; expression of recombinantnaphthalene dioxygenase (NDO) in anengineered bacterial strain for the production ofindigo from glucose; and target-specificbiodegradation of environmental pollutants.

Enantioselectivity of the dioxygenase: Thefine chemicals, natural products, pharmaceutical

intermediates, and biologically active compoundscan be prepeared using Dioxygenase-derivedchiral metabolites (Fig.2). The use ofenzymatically formed cis-diols in enantioselectivesynthesis has been the subject of acomprehensive review (14, 15) a wide range ofcyclitols, conduritols, conduramines, inositols,heteroatom carbohydrates, alkaloids, and avariety of natural products were syntheticdesigned via dioxygenase-catalyzed cis-dihydroxyation and their application inasymmetric methodology for the synthesis of thecis-dihydrodiols of dictamnine and 4-chlorofuroquinoline yielded phenolic derivativesfrom which a range of furoquinoline alkaloidswere synthesized (16). A summary of recentprogress in the synthesis of morphine alkaloidsincluded the use of several metabolites derivedvia dioxygenase biocatalysis. Cyclohexadienecis-dihydrodiols of phenethyl bromide andbromobenzene, as well as 3-bromocatechol(produced by a strain overexpressingTDO andDDH), have been employed as synthons in twoseparated synthetic strategies to produceadvanced intermediates for the synthesis ofmorphine alkaloids (17). The biooxidation of 4-bromoanisole by recombinant E. coli expressingTDO and DDH yielded p-methoxybromocatechol.This functionalized catechol was coupled withtrimethoxyphenylacetylene in convergentsyntheses of combretastatins A-1 and B-1,members of a class of oxygenated naturalproducts with potent cytotoxic activity (18). Anefficient chemoenzymatic synthesis ofstrawberry furanone (4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one), a naturally occurring flavorcompound, was enabled through directedevolution of TDO and tetrachlorobenzenedioxygenase operons that yielded improvedenzymes for the conversion of p-xylene to therequisite diol synthon, cis-1,2-dihydroxy-3,6-dimethyl-3,5-cyclohexadiene (19).

Indinavir production: Cis-(1S)-amino-(2R)-indanol formation as a result of biocatalyticproduction of enantiopure (-)-cis-(1S, 2R)-indandiol, which is a precursor for Merck’s HIV-



1 protease inhibitor Indinivir Sulfate (Crixivan)(20). Pseudomonas putida F39/D or E. coliharbouring tolune dioxygenase (TDO) revealedthat wild-type TDO oxidized indene to (-)-cis-(1S,2R)- indandiol (~30% ee) and (1R)-indenol asthe main products, with traces of 1-indenoneformed ( 21, 22). As a result of indene conversionby wild-type P. putida F1 about 98% of (-)-cis-(1S, 2R)-indandiol was obtained (20), also (-)-cis-(1S, 2R)-indandiol was obtained ascoexpression of dihydrodiol dehydrogenase(DDH) together with Tolune dioxygenase in E.coli (22). As a result of kinetic resolution catalyzedby DDH that is selective for the undesired (þ)-cis-(1R, enantiopurity of (-)-cis-(1S, 2R)-indandiolwas increased at the expense of total indandiolyield (22). Directed evolution was used to selectthe variants of TDO that produced. The reducedamounts of the indene by-products 1- indenol and1-indenone, while maintaining high (-)-cis-(1S,2R)-indandiol enantiopurity was selected invariants of TDO using directed evolution (23).The variants that produced significantly more cis-indandiol relative to the undesired by-productindenol were obtained after three rounds ofmutagenesis. To favor the production of theundesired (+)-cis-indandiol enantiomer, thestereoselectivity was altered (23). The eliminationof indene formation of indene by-products withthe limited yield about 60% (-)-cis-(1S, 2R)-indandiolwas not achieved either with thesestrategies or the application of oxygenases fromRhodococcus strains (24). TDO-catalyzedenantioselective monohydroxylation of 2-indanolto (-)-cis-(1S, 2R)-indandiol is the alternativeroute to the vicinal aminoindanol. Preparation ofchiral 1-hydroxy-2-substituted indanintermediates by this reaction is the basis for thisprocess (25). P. putida strains UV-4 (26) and F39/D expressing TDO oxidized 2-indenol to (-)-cis-(1S, 2R)-indandiol in >98% ee and >85% yield;minor products included trans-1,2-indandiol(<15%) and 2-hydroxy-1-indanone (<2%) (27).

Indigo production: E. coli strain expressingNDO from Pseudomonas sp. NCIB 9816-4 wasfor the first time discovered that it can oxidaize

of indole to indigo was first shown in recombinant(28). NDO oxidized indole to an unstable cis-dihydroindolediol that dehydrates to indoxyl, andsubsequently undergoes spontaneous oxidationto indigo. Dioxygenases catalyze this reactionand by colorimetric test has been widely utilizedfor detection and isolation of strains expressingmono- and dioxygenases, and in screening formutants of these strains (Fig. 3). Commercialinterest in the reaction led Genencor Internationalto genetically engineer a cost-competitive,multistep pathway for the production of indigofrom glucose in E. coli (29). The process forindigo production was based on a recombinantE. coli strain in which the tryptophan pathway wasmodified to allow a high level of indole productionand cloned NDO from P. putida was expressed(29). Numerous modifications were made to thestrain to improve metabolite flux, eliminate theformation of the by-product isatin, and ultimatelyincrease the production of indigo to levelsexceeding 18 g/L. Despite the technical successof the process, the commercial production ofindigo has not been implemented at an industrialscale.

Hamdy A. Hassan

Fig. 3. Production of Indigo using Naphthalenedioxygenase from Indole


Dioxygenase and new biodegradationpathways: The engineering new pathways forthe degradation of recalcitrant compounds havebeen used by aromatic hydrocarbondioxygenases. Cloning of Genes encoding TDOfrom P. putida F1 and cytochrome P450cam intothe genome of Deinococcus radiodurans, in thepresence of high levels of radiation thisrecombinant strain was able to degrade tolueneand related aromatic hydrocarbons (30). Anengineered strain Deinococcus radiodurans hasobtained as results of cloning and expression ofmercury resistance gene (merA) together withthe TDO genes, this strain has the ability toremediate mixed radioactive waste containingaromatic hydrocarbon pollutants and the heavymetal mercury. Genetic engineering was usedto modify many bacterial isolates, that have theability to degrade few number of aromatichydrocarbon pollutants to increase the ability ofthese isolates to degrade a broad range ofaromatic pollutants. When a constructedcassette, carrying genes for the conversion ofstyrene to phenylacetate, was introduced intoP. putida F1 carrying the TOL plasmid, theengineered strain was capable of growth on anexpanded range of aromatic hydrocarbons,including benzene, toluene, ethylbenzene, m-xylene, p-xylene, and styrene (31). The preferredgene cassette, which harbour the interestedgenes system was applied to a mini-transposonto increase the expression of the function genescaste and eliminate the undesirable expressionof horizontal transfer among bacteria in theenvironment through genetic engineering. Sucha strain could prove useful in the bioremediationof low molecular weight aromatic hydrocarbonpollution.

ConclusionMulticomponent aromatic-ring-hydroxy-

lating dioxygenases are class of enzymes holdssignificant promise for oxidize aromatichydrocarbons to vicinal arene cis-diols are ofparamount importance in providing the scientificfoundations necessary for the development of

bioremediation technology and in greenchemistry by the ability of many of these enzymesto form pharmaceutical compounds in highenantiomeric purity are of great interest. Toachieve this target either by identification anenzyme that oxidizes aromatic compoundsubstrate to produce a specific product, theimportant thing firstly one can attempt to isolatea new bacterial strain with the desired ability,secondly screen the large number of well-characterized dioxygenases that are currentlyavailable, thirdly modify an available enzymeknown to have activity by random mutagenesismethods (13, 32, 33) or by rational design, takingadvantage of the growing number of availabledioxygenase crystal structures (34, 35, 36).Finally, If you did not need to isolate andcharacterize the host bacterium, you can screenby metagenomic libraries (37, 38), especially withsamples from diverse environments, may allowthe identification of new dioxygenases with usefulactivities. Many steps are required to developviable and economical commercial processes,once an enzyme with the desired selectivity isobtained. Efficiency can be realized through highprotein expression coupled with processdevelopment. Overall productivity may also beincreased by improving the activity orthermostability of the enzyme, or by reducingproduct inhibition. To get more product anddetermine which material will be used eitherpurified enzymes or whole-cells, the well-designed process must take into considerationtwo things, firstly the multicomponent enzymeand the NADH requirement

AcknowledgementsThe author is grateful to the Science and

Technological Development Fund (STDF)Government of Egypt for financial support to carryout this work as a part of project 46.

References1. Kirk, T.K. and Farrell, R.L. (1987).

Enzymatic “combustion”: the microbialdegradation of lignin. Annu Rev Microbiol41: 465-505



2. Gibson, D.T. and Parales, R.E. (2000)Aromatic hydrocarbon dioxygenases inenvironmental biotechnology. Curr OpinBiotechnol 11: 236-243

3. Pieper, D. H., González, B., Cámara, B.,Pérez-Pantoja, D. and Reineke, W. (2009).Aerobic degradation of chloroaromatics inHydrocarbons, oils and lipids (K. N. Timmis,ed). Vol. 2 Microbiology of utilization ofhydrocarbons, oils and lipids, SpringerVerlag

4. Yuji Ashikawa, Zui Fujimoto, Yusuke Usami,Kengo Inoue, Haruko Noguchi, HisakazuYamane and Hideaki Nojiri (2012).Structural insight into the substrate- anddioxygen-binding manner in the catalyticcycle of rieske nonheme iron oxygenasesystem, carbazole 1,9a-dioxygenase BMCStructural Biology 12:15

5. Gibson, D.T., Koch, J.R. and Kallio, R.E.(1968). Oxidative degradation of aromatichydrocarbons by microorganisms. 1.Enzymatic formation of catechol frombenzene. Biochemistry 7: 2653-2662.

6. Reiner, A.M. (1972). Purification andproperties of the catechol-forming enzyme3,5-cyclohexadiene-1,2-diol-1-carboxylicacid (NAD+) oxidoreductase (decar-boxylating). J Biol Chem 247: 4960-4965.

7. Bundy, B.M., Campbell, A.L. and Neidle,E.L. (1998). Similarities between theantABC-encoded anthranilate dioxygenaseand the benABC-encoded benzoatedioxygenase of Acinetobacter sp. strainADP1. J Bacteriol 180: 4466-4474.

8. Farrow, J.M. and Pesci, E.C. (2007). Twodistinct pathways supply anthranilate as aprecursor of the Pseudomonas quinolonesignal. J Bacteriol 189: 3425-3433.

9. Werlen, C., Kohler, H. P. and van der Meer,J. R. (1996). The broad substratechlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase

of Pseudomonas sp. strain P51 are linkedevolutionarily to the enzymes for benzeneand toluene degradation. J Biol Chem 271,4009-4016.

10. Beil, S., Mason, J. R., Timmis, K. N. andPieper, D. H. (1998). Identification ofchlorobenzene dioxygenase sequenceelements involved in dechlorination of1,2,4,5-tetrachlorobenzene. J Bacteriol180, 5520-5528.

11. Parales, J. V., Parales, R. E., Resnick, S.M. and Gibson, D. T. (1998). Enzymespecificity of 2-nitrotoluene 2,3-dioxygenase from Pseudomonas sp. strainJS42 is determined by the C-terminal regionof the alpha subunit of the oxygenasecomponent. J Bacteriol 180, 1194-1199.

12. Nam, J. W., Nojiri, H., Yoshida, T., Habe,H., Yamane, H. and Omori, T. (2001). Newclassification system for oxygenasecomponents involved in ring-hydroxylatingoxygenations. Biosci Biotechnol Biochem65, 254-263.

13. Zhao, H., Chockalingam, K. and Chen, Z.(2002). Directed evolution of enzymes andpathways for industrial biocatalysis, Curr.Opin. Biotechnol., 13:104–110.

14. Hudlicky, T., Gonzalez, D. and Gibson, D.T.(1999). Enzymatic dihydroxylation ofaromatics in enantioselective synthesis:expanding asymmetric methodology,Aldrichimica Acta, 32: 35–62

15. Eduardo Busto, Vicente Gotor-Fernándezand Vicente Gotor (2012) AsymmetricChemoenzymatic Synthesis of RamatrobanUsing Lipases and Oxidoreductases J. Org.Chem. 77, 4842

16. Boyd, D.R., Sharma, N.D., O’Dowd, C.R.,Carroll, J.G., Loke, P.L. and Allen, C.C.R.(2005). cis- Dihydrodiol, arene oxide andphenol metabolites of dictamnine: keyintermediates in the biodegradation andbiosynthesis of furoquinoline alkaloids,

Hamdy A. Hassan


Chem. Commun., 31: 3989–399

17. Zezula, J. and Hudlicky, T. (2005). Recentprogress in the synthesis of morphinealkaloids, Synlett, 3, 388–405.

18. Bui, V.P., Hudlicky, T., Hansen, T.V. andStenstrom, Y. (2002). Direct biooxidation ofarenes to corresponding catechols with E.oli JM109(pDTG602). Application tosynthesis of combretastatins A-1 and B-1,Tetrahedron Lett., 43:2839–284.

19. Newman, L.M., Garcia, H., Hudlicky, T., andSelifonov, S.A. (2004). Directed evolutionof the dioxygenase complex for thesynthesis of furanone flavor compounds,Tetrahedron, 60, 729–734.

20. Connors, N., Prevoznak, R., Chartrain, M.,Reddy, J., Singhvi, R., Patel, Z., Olewinski,R., Salmon, P., Wilson, J. and Greasham,R. (1997). Conversion of indene to cis-(1S,2R)-indandiol by mutants of Pseudomonasputida F1, J. Ind. Microbiol. Biotechnol., 18:353–359.

21. Wackett, L.P., Kwart, L.D. and Gibson, D.T.(1988). Benzylic monooxygenationcatalyzed by toluene dioxygenase fromPseudomonas putida, Biochemistry, 27:1360–1367.

22. Reddy, J., Lee, C., Neeper, M., Greasham,R. and Zhang, J. (1999). Development of abioconversion process for production of cis-1S, 2R-indandiol from indene byrecombinant Escherichia coli constructs,Appl. Microbiol. Biotechnol., 51: 614–620.

23. Zhang, N., Stewart, B.G., Moore, J.C.,Greasham, R.L., Robinson, D.K., Buckland,B.C. and Lee, C. (2000). Directed evolutionof toluene dioxygenase from Pseudomonasputida for improved selectivity toward cis-indandiol during indene bioconversion,Metab. Eng., 2: 339–348.

24. O’Brien, X.M., Parker, J.A., Lessard, P.A.and Sinskey, A.J. (2002). Engineering an

indene bioconversion process for theproduction of cis-aminoindanol: a modelsystem for the production of chiral synthons,Appl. Microbiol. Biotechnol., 59, 389–399

25. Blacker, A.J., Boyd, D.R., Dalton, H. andBowers, N. (1996). Preparation of hydroxylcompounds by conversion with dioxy-genase. WO 96/37628, May 20.

26. B o y d , D . R . , S h a r m a , N . D . , B o w e r s ,N.I.,Goodrich, P.A.,Groocock, M.R., Blaker,A.J., Clarke, D.A., Howard, T. and alton, H.(1996). Stereoselective dioxygenase-catalyzed benzylic hydroxylationat prochiralmethylene groups in the chemoenzymaticsynthesis of enantiopure vicinalamino-indanols, Tetrahedron Asymmetry, 7: 1559–1562.

27. Lakshman, M.K., Chaturvedi, S., Zajc, B.,Gibson, D.T. and Resnick, S.M. (1998). Ageneral chemoenzymatic synthesis ofenantiopure cis b-amino alcohols frommicrobially derived cis-glycols, Synthesis,9:1352–1356.

28. Ensley, B.D., Ratzkin, B.J., Osslund, T.D.,Simon, M.J., Wackett, L.P. and Gibson, D.T.(1983) Expression of naphthalene oxidationgenes in Escherichia coli results in thebiosynthesis of indigo, Science, 222:167–169.

29. Berry, A., Dodge, T.C., Pepsin, M. andWeyler, W. (2002). Application of metabolicengineering to improve both the productionand use of biotech indigo, J. Ind. Microbiol.Biotechnol., 28:127–133.

30. Lange, C.C., Wackett, L.P., Minton, K.W.and Daly, M.J. (1998). Engineering arecombinant Deinococcus radiodurans fororganopollutant degradation in radioactivemixed waste environments, Nat.Biotechnol., 16, 929–933.

31. Lorenzo, P., Alonso, S., Velasco, A., Diaz,E., Garcia, J.L. and Perera, J. (2003)Design of catabolic cassettes for styrene



biodegradation, Antonie VanLeeuwenhoek., 84:17–24.

32. Cirino, P.C. and Arnold, F.H. (2002). Proteinengineering of oxygenases for biocatalysis,Curr. Opin. Chem. Biol., 6, 130–135.

33. Jaeger, K.E. and Eggert, T. (2004).Enantioselective biocatalysis optimized bydirected evolution, Curr. Opin. Biotechnol.,15, 305–313.

34. Kauppi, B., Lee, K., Carredano, E., Parales,R.E., Gibson, D.T., Eklund, H. andRamaswamy, S. (1998). Structure of anaromatic ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase, Structure,6:571–586.

35. Furusawa, Y., Nagarajan, V., Tanokura, M.,Masai, E., Fukuda, M. and Senda, T. (2004).Crystal structure of the terminal oxygenase

of biphenyl dioxygenase from Rhodococcussp. strain RHA1, J. Mol. Biol., 342: 1041–1052.

36. Martins, B.M., Svetlitchnaia, T. and Dobbek,H. (2005). 2-Oxoquinoline 8-monooxy-genase oxygenase component: active sitemodulation by Rieske-[2Fe-2S] centeroxidation/reduction, Structure, 13: 817–824.

37. Daniel, R. (2004). The soil metagenome arich resource for the discovery of novelnatural products, Curr. Opin. Biotechnol.,15: 199–204.

38. Lorenzo, P., Liebeton, K., Niehaus, F. andEck, J. (2002). Screening for novelenzymes for biocatalytic processes:accessing the metagenome as a resourceof novel functional sequence space, Curr.Opin. Biotechnol., 13: 572–577.

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