3.2.1 Morphine Alkaloid Biosynthesis 3423.2.2 Microbial Metabolism of Morphine Alkaloids 3443.2.3 Transformations of Morphine Alkaloids by Pseudomonas putida M10 3463.2.4 Biological Production of Hydromorphone and Hydrocodone 3503.2.5 Microbial Transformation of Heroin 351
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1 Introduction
Plants have the ability to produce tens ofthousands of highly complex secondary me-tabolites to assist their survival in the environ-ment, many of which protect the plant frompredators. Man has exploited these com-pounds of self-defence as sources of medicinalagents, poisons, and potions since time imme-morial. Throughout the world different com-munities have discovered plants with phar-macological properties, and many useful drugshave their origins in indigenous ethnopharma-cologies. Some notable examples include: theroots of the mandrake plant, known for theirsedative properties from the time of HIPPO-CRATES (ca. 400 BC) and also used as a deadlypoison during Elizabethan times (MANN,1989); the leaves of the coca plant, which werechewed as an aid to stamina and as part of ce-remonies in South America over 5000 yearsago (VAN DYKE and BYCK, 1982); and plantswhose hallucinogenic properties were used inthe preparation of “magic potions” by the Az-tec Indians. The compounds responsible forthese physiological effects in man were isolat-ed during the 19th and early 20th centuries andwere identified as scopolamine, cocaine, andamides of lysergic acid, respectively. SOCRA-TES’ death in 399 BC was the result of con-sumption of hemlock (Conium maculatum)which contains the alkaloid coniine (1) (Fig. 1;HENDRICKSON et al., 1970), while CLEOPATRA
used extracts from Egyptian henbane(Hyoscyamus muticus) during the last centuryBC to dilate her pupils and increase her beau-ty. Likewise, medieval European women usedextracts of deadly nightshade (Atropa bella-donna) in their beauty preparations, hence thename bella donna, “fair lady”.
Other historical uses include extracts fromthe bark of Cinchona officinalis which havebeen employed as antimalarials. Extracts de-rived from the opium poppy Papaver somni-ferum comprise another group of importantpharmacologically active compounds whichpossess powerful analgesic properties. It hasbeen reported that extracts of the milky latexmaterial that exudes from the cut unripe seedcapsule of the opium poppy were used by theearly Egyptians for medicinal purposes; how-
ever, results from a recent examination of ma-terials from the tomb of the Royal ArchitectKHA seem to refute earlier observations (BIS-SET et al., 1994). It was not until the 19th centu-ry that the active compounds, alkaloids, wereisolated from the opium. Morphine was alsothe first alkaloid to be identified and crystal-lized by the chemist SERTÜRNER in 1805. Thiswas a significant achievement as not only wasit the first time that a nitrogenous base hadbeen isolated from a biological source, but itwas also the first time that such a substancehad been shown to be intrinsically basic. Thisfinding formed the basis of one of the earliestdefinitions of an alkaloid which was attributedto the pharmacist W. MEISSNER (HESSE, 1981;PELLETIER, 1983). The two authors, HESSE
(1981) and PELLETIER (1983), differ on the ex-act date of the coining of the term (1818 and1819 being cited, respectively) and the deriva-tion, but the general meaning was taken to bean “alkali like” compound of plant origin, or asBENTLEY (1954) interpreted, a “vegetable al-kali”. This was extended by WINTERSTEIN andTIET (1910) to include a four part definitionstating a “true alkaloid” can be characterizedby: (1) the possession of a nitrogen atom aspart of a heterocyclic system; (2) a complexmolecular structure; (3) significant pharmaco-logical properties; (4) its origin from the plantkingdom (cited in PELLETIER, 1983).
The majority of alkaloids fit this four partdefinition; however, a number of exceptionsexist.The compounds samandarine (2) (Fig. 1),samandarone, and cycloneosamandarine, iso-lated from the skin glands of the European firesalamander (Salamandra maculosa Laurenti)all exhibit the usual properties of an alkaloidsubstance, but do not fit the definition of a“true alkaloid” owing to their animal origin.There are numerous examples of alkaloids fur-nished by animal, including batrachotoxinin A(3), a steroidal alkaloid from the Colombianarrow-poison frog (Phyllobates aurotaenia),bufotenine (4), a tryptamine-type alkaloidfrom the common European toad, (P)-deoxy-nuphradine and (P)-castoramine (5) from theCanadian beaver (Castor fiber L.), and musco-pyridine (6) from the scent gland of the muskdeer (Moschus moschiferus). Alkaloids havealso been identified from arthropod, bacterial,and fungal origins. For example, the quinazole
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1 Introduction 329
alkaloids, glomerine (7a) and homoglomerine(7b) discharged from the dorsal glands of theEuropean millipede (Glomeris marginata), thedeep-blue colored alkaloid pyocyanine (8),isolated from the bacterium Pseudomonas ae-ruginosa, and agroclavine (9), produced by thefungi Claviceps purpurea and Aspergillus fu-migatus. Other examples of alkaloids existwhich also do not adhere to the criteria stipu-lated in the four part definition of an alkaloid.For example, the alkaloids colchicine (10) (au-tumn crocus, Colchicum autumnale L.) andmescaline (11) (Lophophora williamsii) donot possess nitrogen as part of a heterocyclicsystem. Also colchicine (10) is essentially neu-
tral and, therefore, does not conform to theoriginal definition of an alkaloid. PELLETIER
(1983), however, provides a reasonable sum-mary of an alkaloid’s properties as being an“alicyclic compound containing nitrogen in anegative oxidation state which is of limited dis-tribution among living organisms”. Over10000 compounds fall within this definition(SOUTHON and BUCKINGHAM, 1989) and newalkaloids are continually being reported fromvarious sources. These represent approximate-ly 20% of all known natural products; howev-er, only about 30 of these with biological activ-ity are commercialized (FARNSWORTH, 1990).
Fig. 1. See text.
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Unlike any other group of compounds thealkaloids exhibit a vast array of skeletal typesand are classified accordingly. A typical exam-ple is the scheme used by HESSE (1981), whodescribes 11 classes of heterocyclic alkaloids,differentiating by the nature of the carbonskeleton, e.g., the pyrrolidine and isoquinolinealkaloids. The majority of alkaloids are aminoacid-derived, although terpenes, steroids, pu-rines, and nicotinic acid can also act as buildingblocks of, e.g., aconitine, solanidine, caffeine,and nicotine, respectively. If the anabolic routeof an alkaloid is known, this can be used toclassify the compound (DALTON, 1979). Thetropane, and pyrrolidine alkaloids, for in-stance, are all derived from ornithine, a deriva-tive of arginine, and thus grouped togetherunder this scheme.
Alkaloids have provided a wealth of phar-macologically active compounds; approxi-mately 25% of the drugs used today are ofplant origin. These are administered either aspure compounds or as extracts and have oftenserved as model structures for synthetic drugs,e.g., atropine (13) for tropicamide, quinine forchloroquinine, and cocaine (12) (Fig. 2) forprocaine (KUTCHAN, l995). Screening of plantextracts for pharmacologically active com-pounds still continues and results in new drugdiscoveries; recent examples include the anti-cancer drugs taxol from the western yew, Taxusbrevifolia, and camptothecin from Camptothe-ca acuminata. Alkaloids are generally regard-ed as speciality chemicals; approximately300–500 metric tons of quinine and quinidineare produced each year; ajmalicine (98) pro-duction amounts to about 3600 kg, while com-pounds like vincristine (94) and vinblastine(95) (Fig. 21) are produced in the kilogramrange. The annual market value of the majoralkaloids has been estimated to be in the rangeof several hundred million dollars (VER-POORTE et al., 1993).
The important pharmacological activity ofmany alkaloids has spurred chemists to makemany derivatives of these natural compounds.The chemical preparation of such semisynthet-ic alkaloids has resulted in the production ofdrugs with improved properties, such as theaddition of a 14-hydroxy group to the mor-phine alkaloid structure which has been foundto dramatically increase potency (JOHNSON
and MILNE, 1981). However, the synthesis ofsuch compounds is often difficult to achieve ona commercial scale due to the chemical com-plexity of the starting material, cost, and envi-ronmental issues, in addition to the precursorsbeing in limited supply. Biotransformationscan offer a number of advantages over con-ventional chemical processes.The specificity ofenzyme-catalyzed reactions, e.g., allows thestereospecific transformation of defined func-tional groups. However, biotransformations ofalkaloids, unlike their steroid counterparts,have yet to meet their potential on an industri-
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Fig. 2. Examples of tropane alkaloids.
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1 Introduction 331
al scale. This is in part due to the lack of suit-able enzymes and partly because no alkaloid-based drug commands a significant share ofthe therapeutic market, unlike the steroids.
The rate at which new drugs derived fromnatural products are entering the therapeuticmarket has declined significantly over recentperiods in contrast to synthetic molecules, pos-sibly due to the difficulty of modifying theseoften complex chemicals for the developmentof new drugs and the difficulty of producingthese natural products in a pure form. The dif-ficulties associated with the development ofnew drugs are being addressed by the ever in-creasing interplay of chemistry and biology.Undoubtedly combinatorial approaches(AMATO, 1992) and genetic engineering willplay an important role in the development ofnew drugs.
The use of recombinant DNA technology isbeginning to have substantial impact on bio-transformation processes, resulting in the de-velopment of new approaches using biologicalsystems. Recent advances in the understandingof the genetic and biochemical basis of alka-loid biosynthetic pathways are now beginningto make biotransformations of complex alka-loid molecules more plausible. The expressionof plant enzymes, which are often present atvery low levels in the plant, in heterologoushosts such as bacteria allows detailed examina-tion of mechanisms of reaction which are oftenunknown in synthetic organic chemistry. It ispossible to add to the genetic repertoire of aplant by incorporating genes from other spe-cies allowing the possibility of producingunique compounds with potential biotechno-logical applications. Microorganisms havebeen used for the large-scale production ofhigh-value chemicals for many years, and theuse of microbial processes to make analogs ofnaturally occurring alkaloids is achievable. It isnow possible to assemble hybrid transforma-tion pathways in microbes using structuralgenes cloned from different organisms whichmediate enzymic processes which are not in-digenous to the host organism. These “patch-work” pathways can have the advantage of re-moving unwanted side reactions, they allowthe possibility of increasing the activity of acell by altering regulatory processes, and avoidthe need to supply expensive exogeneous co-
factors. Furthermore, it is now theoreticallypossible to manipulate alkaloid biosyntheticpathways to improve yields and to extendpathways to synthesize new bioactive mole-cules. Metabolic engineering of plants offersthe capability of altering the pattern of alka-loid accumulation in the plant; in addition, theability to house and express recombinantgenes in plants from other organisms offersthe potential of both extending pathways andallowing the biological synthesis of semi-synthetic derivatives. It is now possible to de-sign strategies to alter the metabolic flux in avariety of organisms, such as the introductionof extra copies of genes encoding enzymeswhich form bottlenecks in pathways affords away to attain increases in yields of plant secon-dary products, or more globally through theexpression of one or more regulatory genes(for reviews see BAILEY, 1991; NESSLER, 1994;HUTCHINSON, 1994; KUTCHAN, 1995). Due totheir complex structures, alkaloids are stillmost efficiently produced by the plant and thefuture success of metabolic engineering ofplant secondary products is dependent on hav-ing a good understanding of the biochemistryand regulation of the pathways under consid-eration.
Plant cell culture has been invaluable as ameans of providing suitable biomass for theelucidation of pathways for secondary metabo-lites, particularly for alkaloid synthesis. Cellculture has also been examined for biotrans-formation purposes (reviewed by VERPOORTE
et al., 1993) and extensively investigated as ameans of producing plant secondary productson a large scale. Unfortunately, the level andmanner of production of alkaloids in plantsdoes not necessarily correlate with productionin cell cultures. The use of plant cell culturesfor biotransformations of alkaloids will not beconsidered in detail this chapter. The purposeof this review is to introduce some of thenewer technologies which are beginning tomake an impact on the area of alkaloid trans-formations. It also aims to introduce some ofthe more recent developments in the biochem-istry and genetic understanding of biosynthet-ic and catabolic routes of some of the morepharmacologically important alkaloids in dif-ferent organisms, without which the rationaldesign of any recombinant alkaloid biotrans-
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formation processes would not be feasible.Thealkaloids discussed include the tropane alka-loids, the benzylisoquinoline alkaloids, thebenzophenanthridine alkaloids, the morphinanalkaloids, and the monoterpenoid indole alka-loids.
2 Tropane AlkaloidsThe tropane alkaloids occur in the Solana-
ceae family, but they are also found in theplant families Erythroxylaceae, Convolvula-ceae, Proteaceae, and Rhizophoraceae. Theircommon structural element is the azabicyc-lo[3.2.1]octane system, and over 150 tropanealkaloids have been isolated. The 3-hydroxyaromatie ester derivatives form the parent al-kaloids, examples of which include cocaine(12) (Erythroxylon coca, coca plant), hyoscy-amine (13), (Hyoscyamus niger, henbane),atropine (13) (Atropa belladonna, deadlynightshade), and scopolamine (14) (Scopolacarniolica) (Fig. 2). It appears that in mostcases atropine (13) is formed by racemizationof hyoscyamine (13) during extraction.
Long before the elucidation of their struc-tures, the pharmacological properties of sever-al tropane alkaloids were exploited. Atropine(13), which typifies the action of tropane alka-loids, causes antagonism to muscarine recep-tors (parasympathetic inhibition) (CORDELL,1981).
These receptors are responsible for slowingof the heart rate, vasodilation, dilation of thepupil, and stimulation of secretions. The heartrate altering properties of atropine (13) haveled to its use in the initial treatment of myocar-dial infraction. Tropane alkaloids have alsobeen used to treat peptic ulcers, prevent mo-tion sickness, and as components of pre-an-esthetic drugs. Cocaine (12) is perhaps the best known of all the tropane alkaloids mainlybecause of its use as an illicit drug; it is a pow-erful central nervous system stimulant and ad-renergic blocking agent, and its hydrochloridesalt has been used as a local and surface anes-thetic in face, eye, nose, and throat surgery(GERALD, 1981). The function of cocaine (12)in leaves of the coca plant was unknown until a
recent study suggested that the alkaloid has in-secticidal properties at naturally occurringconcentrations due to potentiation of insectoctopaminergic neurotransmission (NATHAN-SON et al., 1993).
2.1 Tropane Alkaloid Biosynthesis
The biosynthetic pathways for the tropanealkaloids have been studied in considerabledetail and are associated with nicotine biosyn-thesis, since the N-methyl-D1-pyrrolinium cat-ion (15) is a precursor to both classes of alka-loids. The formation of the tropane nucleusfrom ornithine and acetoacetate was first in-vestigated in plants using radioactive tracers aslong ago as 1954, but it was not until the early1980s that a biosynthetic scheme was finallyelucidated in Erythroxylon coca (Fig. 3; LEETE,1983). The pathway for biosynthesis of hyos-cyamine (13) and scopolaminc (14) is quitecomplex, since not only is an acetone unit re-quired for the formation of tropinol (19), but asecond converging pathway is necessary forthe conversion of phenylalanine (20) to tropicacid (23) (Fig. 4). Recent work with root cul-tures of Datura stramonium, suggests that hy-grine (17) is not an intermediate, but an offshoot from the main pathway (ROBINS et al.,1997). The biosynthesis of cocaine (12) is simi-lar to that of hyoscyamine (13). The N-methyl-ation and cyclization of ornithine-derived pu-trescine gives the N-methyl-D1-pyrroliniumcation (15), which condenses with acetoacetyl-CoA (16). Methylation of the free carboxylategroup followed by ring closure, reduction ofthe ketone group, and benzoylation results inthe formation of cocaine (12). The benzoic ac-id is derived from phenylalanine (20).
The molecular biology of tropane alkaloidsynthesis is being studied extensively andholds considerable potential for alkaloid bio-transformations. An elegant example is theconstruction of a transgenic species of Atropabelladonna that was able to accumulate theimportant pharmaceutical scopolamine (14)instead of hyoscyamine (13) (YUN et al., 1992).The final two steps in the pathway for the bio-synthesis of scopolamine (14) (Fig. 5) are cata-lyzed by 2-oxoglutarate-dependent hyoscy-amine 6b-hydroxylase (hyoscyamine[6b]-di-
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2 Tropane Alkaloids 333
oxygenase; EC 1.14.11.11). This enzyme firsthydroxylates hyoscyamine in the 6b-positionof the tropane ring (24), which is followed byepoxidation. The use of purified 6b-hydroxy-lase from root cultures of Hyoscyamus niger
showed that catalysis of these two steps car-ried out was by the same enzyme (HASHIMOTO
et al., 1987). Analysis of key enzymes of meta-bolic pathways at the molecular genetic levelassists clarification of complex biochemicalmechanisms, and hydrolysis and epoxidationof the tropane ring was later confirmed un-equivocally by molecular cloning and expres-sion of the structural gene of the 6b-hydroxyl-ase in a heterologous host (MATSUDA et al.,1991; HASHIMOTO et al., 1993b); A. belladonnaaccumulates hyoscyamine (13) instead of sco-polamine (14) because it lacks the 6b-hydrox-ylase. The cDNA encoding the 6b-hydroxylasefrom H. niger was transferred into Agrobacte-rium tumefaciens and introduced into A. bella-donna.The regenerated transgenic plants werefound to contain elevaled levels of scopol-amine (14) (YUN et al., 1992). The change in
Fig. 3. Biosynthesis of tropane alkaloids (ROBINS
et al., 1994).
Fig. 4. Biosynthesis of tropic acid (ROBINS et al.,1994).
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alkaloid composition in the transgenic A. bel-ladonna was considerable, with scopolamine(14) being almost the only alkaloid present inthe aerial parts of the plant. It was thus pos-sible to isolate pure scopolamine (14) by re-crystallization of the total alkaloid fraction, in-stead of conventional differential extractionand chromatography.Analysis of expression ofthe 6b-hydroxylase gene by measurements oflevels of mRNA and Western blot analysis ofprotein extracts from various tissues showedthat enzyme expression in scopolamine pro-ducing species of Hyoscyamus was lacking inthe stem or leaves, being localized in the rootsof these plants, and explains why it has notbeen possible to produce these alkaloids in sig-nificant quantities by cell culture (HASHIMOTO
et al., 1991; MATSUDA et al., 1991). HASHIMOTO
et al. (1993b) have now engineered transgenicA. belladonna hairy root cultures that expressthe H. niger gene encoding hyoscyamine 6b-hydroxylase which exhibited up to 5 timeshigher activity. These transgenic roots mayprove to be useful for enhancing scopolamineproductivity in vitro. Recombinant strains ofEscherichia coli expressing the gene encodinghyoscyamine hydroxylase were also capable oftransforming hyoscyamine (13) to scopol-amine (14) (HASHIMOTO et al., 1993a; LAY etal., 1994).
Tropinone reductase acts at a branch pointof biosynthetic pathways leading to a varietyof tropane alkaloids. It is an NADPH-de-pendent enzyme which reduces the 3-ketogroup of tropinone (18) (ROBINS et al., 1994).Two tropinone reductases with different ster-
eospecificities were found in cultured roots ofH. niger (HASHIMOTO et al., 1992).
These two distinct enzyme activities reduc-ed tropinone (18) to 3a-hydroxytropane (19)(tropinol, tropine) and 3b-hydroxytropane(pseudotropinol, c-tropine, pseudotropine),respectively. Marked differences were ob-served between the two reductases in their af-finities for tropinone (18), substrate specificity,and in the effects of amino acid modificationreagents. The cDNA clones for the two tropi-none reductases have been expressed in Es-cherichia coli and sequenced (NAKAJIMA et al.,1993). Preparation of various chimeric formsof these two enzymes led to the identificationof the domain conferring the stereospecificityof the reaction (NAKAJIMA et al., 1994).
These elegant experiments demonstrate akey to future alkaloid biotransformation pro-cesses by the manipulation of biosyntheticroutes in plants with the use of recombinantDNA technology not just for alkaloids but alsoother secondary products. It is becoming pos-sible to design strategies to advantageouslymanipulate the metabolic flux in organisms, orto decrease or increase the production of phy-tochemicals; however, with regard to biotrans-formations, it is the possibility of manipulatingpathways by altering enzyme function by di-rected mutagenesis and extending/altering ex-isting pathways by heterologous gene expres-sion to alter the spectrum of plant alkaloidswhich is particularly challenging and exciting.Although the genetic tools for manipulatingbiosynthetic pathways in plants lag behindthose for prokaryotic organisms, the success of
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Fig. 5. See text.
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scopolamine production in transgenic plantswill, hopefully, encourage interest and furtherdevelopment.
2.2 Microbial Metabolism ofTropane Alkaloids
Microorganisms possess an incredible varie-ty of metabolic pathways which enables themto degrade a plethora of natural and man-made organic compounds. The elucidation ofalkaloid dissimulating pathways has consider-able potential for the identification of bio-transformation routes for new and existingtherapeutic compounds (BRUCE et al., 1995).
The most extensively studied tropane alka-loid, in terms of microbial metabolism is atro-pine (13). Several bacterial species have beenshown to possess an esterase that catalyzes theesterolytic hydrolysis of the atropine molecule,to form tropinol (19) and tropic acid (23) (Fig.6).
The interest in atropine esterase lies in itssimilarity to mammalian serine proteases andits use as a possible model of mammalian chol-inergic receptors. RÖRSCH et al. (1971) re-ported the isolation of a number of Pseudo-monas strains capable of utilizing atropine as asole source of carbon and nitrogen. The atro-pine esterase from one of these strains, Pseu-domonas putida PMBL-1, has been purifiedand extensively characterized (VAN DER
DRIFT, 1983; VAN DER DRIFT et al., 1985a, b,1987). This esterase showed activity with bothenantiomers of hyoscyamine (13), but not withcocaine (12) (RÖRSCH et al., 1971), despite theclose similarity of structure of those com-pounds. NIEMER et al. (1959) and NIEMER andBUCHERER (1961) reported a breakdownroute of atropine (13) by Corynebacterium bel-ladonnae, which involves the formation oftropinol (19) and tropic acid (23) by esteraseaction, followed by dehydrogenation, ringopening, and deamination of the tropane ring(Fig. 6). The first step in their proposed routeof tropine catabolism involves a tropine dehy-drogenase.Activity, however, was only demon-strated in the reverse direction. The step pos-tulated by NIEMER and BUCHERER (1961)which follows tropinone formation involves
2 Tropane Alkaloids 335
ring cleavage and the formation of tropinic ac-id (25), though no enzymes or cofactors wereidentified. Isolation of a picrate derivative ofmethylamine from whole cell incubations withtropinone, indicated that nitrogen debridgingwas taking place. More recent investigationsinto the microbial metabolism of atropineshowed that a strain of Pseudomonas sp.(termed AT3) isolated from the rhizosphere ofatropine plants was able to utilize tropinol (19)as a sole carbon and nitrogen source (LONG etal., 1993). Growth studies revealed a diauxicgrowth pattern. When this organism was sup-plied with atropine (13) and an exogenous ni-trogen source, tropic acid (23) was utilizedduring the first phase of growth and the heter-ocyclic moiety, tropinol (19), was utilized in thesecond. The enzymes responsible for tropinol(19) degradation appeared to be strongly re-pressed during the first phase of growth.Under nitrogen limitation, however, the nitro-gen must be stripped from the tropane ring be-fore growth can occur and under these condi-tions tropinol (19) was utilized in the firstgrowth phase. Pseudomonas sp. AT3 initiatedthe degradation of tropinol (19) by attackingthe nitrogen atom, yielding a dinitrophenyl hy-drazine positive intermediate, identified as 6-hydroxycyclohepta-1,4-dione (28), which wasoxidized by an NADc-dependent dehydrog-enase activity to cyclohepta-1,3,5-trione (29).The subsequent cleavage of this compound re-sulted in the formation of 4,6-dioxoheptanoicacid (succinylacetone) (30) which was, in turn,the substrate for a second hydrolase yieldingsuccinate (31) and acetone(32) (BARTHOLO-MEW et al., 1993, 1996).
BARTHOLOMEW et al. (1995) identified anNADPc-dependent tropinol dehydrogenasein cell-free extracts of Pseudomonas sp. AT3that was induced by growth on atropine (13),tropinol (19) or tropinone (18).The product ofthe reaction was tropinone (18) and the reac-tion was shown to be freely reversible. The de-hydrogenase showed activity only with tropi-nol (19) and nortropinol (27); no activity wasdetected with a number of closely related com-pounds including atropine (13), scopine, pseu-dotropinol, ecgonine (34) (Fig. 7) and 6-hy-droxycyclohepta-1,4-dione (28), which sug-gests thal this inducible enzyme is involved inthe metabolism of tropinol (19) in Pseudomo-
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Fig. 6. Proposed pathway for the degradation of atropine (13) by Pseudomonas sp.AT3 (BARTHOLOMEW etal., 1996). ----P Pathway proposed by NIEMER and BUCHERER (1959) for C. belladonna.
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nas sp. AT3. However, the occurrence of 6-hy-droxycyclohepta-1,4-dione (28) during the me-tabolism of tropinol seemed to dispute this(BARTHOLOMEW et al., 1993).An elegant set ofexperiments with tropinol (19) and pseudo-tropinol labeled with deuterium in the C-3 po-sition provided an answer (BARTHOLOMEW etal., 1995).The labeled alcohol group at C-3 wasshown to remain intact past the point of re-moval which indicated that tropinone (18) isnot an intermediate in the pathway of tropinol(19) metabolism.What then is the role of trop-inol dehydrogenase in the metabolism of trop-inol? Tropinone (18) serves as a growth sub-strate for Pseudomonas sp. AT3 and it is likelyto be encountered in nature, along with atro-pine (13) and tropinol (19), as it is an interme-diate in the biosynthesis of the tropane alka-loids in plants (LANDGREBE and LEETE, 1990).A mutant strain of Pseudomonas sp. AT3blocked in 6-hydroxycyclohepta-1,4-dione de-hydrogenase activity was grown on tropinone;this resulted in the accumulation of 6-hydroxy-cyclohepta-1,4-dione (28), an indication thattropinone (18) is metabolized via the sameroute as tropinol (19) and that its keto group isreduced in the process. Thus, the tropinol de-hydrogenase may function primarily as a re-ductase in order to channel tropinone (18) andnortropinone into the pathway of tropinol (19)metabolism in Pseudomonas sp. AT3.
The elucidation of the pathway for microbi-al metabolism of the related alkaloid cocaine(12) has proven to be slightly more elusive. Astrain of Pseudomonas maltophilia (termedMB11L) was isolated from samples taken inand around a pharmaceutical company thatprocesses cocaine. P. maltophilia MB11L wascapable of utilizing cocaine (12) as its solesource of nitrogen and carbon for growth. Thebacterium possessed an inducible cocaineesterase which converted cocaine (12) to ecgo-nine methyl ester (33) (Fig. 7), and benzoic ac-id. Both degradation products supportedgrowth of P. maltophilia MB11L, although on-ly cocaine induced high activities of the co-caine esterase (BRITT et al., 1992). Benzoic ac-id was further metabolized via catechol andthe 3-oxoadipate pathway; however, the path-way for the metabolism of ecgonine methyl es-ter (33) was not further elucidated.
Fig. 7. Proposed pathway for the metabolism of co-caine (12) by P. fluorescens MBER and C. acidovor-ans MBLF (LISTER et al., 1995).
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