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Thiamin-dependent enzymes as catalysts in chemoenzymatic syntheses
Biochimica et Biophysica Acta 1385 (1998) 229^243
Review
Thiamin-dependent enzymes as catalysts in chemoenzymatic syntheses Ulrich Scho«rken, Georg A. Sprenger * Institut fu«r Biotechnologie 1, Forschungszentrum Ju«lich GmbH, P.O. Box 1913, D-52425 Ju«lich, Germany Received 18 November 1997; accepted 12 February 1998
Abstract Enzymes are increasingly being used to perform regio- and enantioselective reactions in chemoenzymatic syntheses. To utilize enzymes for unphysiological reactions and to yield novel products, a broad substrate spectrum is desirable. Thiamin diphosphate (ThDP)-dependent enzymes vary in their substrate tolerance from rather strict substrate specificity (phosphoketolases, glyoxylate carboligase) to more permissive enzymes (transketolase, dihydroxyacetone synthase, pyruvate decarboxylase) and therefore differ in their potential to be used as biocatalysts. We give an overview of the known substrate spectra of ThDP-dependent enzymes and present examples of multi-enzyme or chemoenzymatic approaches which involve ThDP-dependent enzymes as biocatalysts to obtain pharmaceutical compounds as ephedrine and glycosidase inhibitors, sex pheromones as exo-brevicomin, 13 C-labeled metabolites, and other intermediates as 1-deoxyxylulose 5-phosphate, a precursor of vitamins and isoprenoids. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Thiamin diphosphate; Transketolase; Pyruvate dehydrogenase; Pyruvate decarboxylase; 1-Deoxyxylulose 5-phosphate
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
230
2.
Speci¢c enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Transketolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Dihydroxyacetone synthase (formaldehyde transketolase) . . . 2.3. 1-Deoxyxylulose-5-phosphate synthase . . . . . . . . . . . . . . . . . 2.4. Pyruvate dehydrogenase and other dehydrogenase complexes 2.5. Pyruvate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Benzoylformate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . 2.7. Miscellaneous ThDP enzymes . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . .
230 230 234 235 236 237 237 239
3.
Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
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* Corresponding author. Fax: +49 (2461) 612710; E-mail: [email protected] 0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 0 7 1 - 5
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U. Scho«rken, G.A. Sprenger / Biochimica et Biophysica Acta 1385 (1998) 229^243
1. Introduction Enzymes are ¢nding increasing acceptance as catalysts in pure and applied chemistry especially for their intrinsic chiral function as they deliver a certain enantiomer at a high enantiomer excess (% ee), combined with a high catalytic e¤ciency. In view of an increasing demand for enantiomerically pure compounds for, e.g., pharmaceutical products, the bene¢ts of enzymes are obvious. Environmental issues are also in favor of enzymes as enzymes help to save on hazardous compounds, organic solvents and other chemical waste. Especially in the ¢eld of carbohydrate chemistry, the inherent multifunctionality of sugars is an enormous task for an organic chemist who has to use a plethora of protective groups in order to prevent unwanted reactions of the hydroxyl, keto, or phosphate groups. A variety of enzymes, mostly lyases and aldolases, have been used so far to synthesize complex sugars, sugar analogues and other biologically important natural compounds [1^ 8]. Thiamin-dependent enzymes have been used for quite a while as catalysts in chemoenzymatic syntheses [3,7^10]. As far as we can tell, the earliest commercial utilization of a thiamin-dependent enzyme, and one of the ¢rst biotransformation processes to be commercialized at all, is the production with whole yeast cells of phenylacetylcarbinol, a precursor of L-ephedrine ([11], Knoll procedure), a process which is in use since the 1930s. It has been established meanwhile [12,13] that the underlying principle of reaction (benzaldehyde plus pyruvate to phenylacetylcarbinol) is being catalyzed by pyruvate decarboxylase. An in-depth treatise of this case is given in this issue by Iding et al. Nowadays, the enzyme which has also attracted a lot of interest in chemoenzymatic syntheses is transketolase from various microorganisms or from plants (e.g. spinach) as it has been used for various carboncarbon (C-C) bonding reactions. A certain drawback, however, has been the availability of commercial enzymes at a good quality, reasonable prices, and a reliable supply from biological sources. With the advent of molecular biology and the use of highly productive recombinant microorganisms, these issues are of lesser importance as it has been shown that enzymes as transketolase from Escherichia coli can be
supplied in amounts surpassing 1 million units easily [14^18]. This should help to increase the acceptance of enzymes for chemoenzymatic syntheses. In most ThDP-dependent reactions the holoenzyme catalyzes a carbon-to-carbon-bond cleavage of the substrate which is followed by a condensation of a portion of the cleaved substrate (associated with ThDP) with an acceptor to form the product. The nature of the acceptor determines whether the enzyme is speci¢c in its reaction or more permissive. For synthetic purposes permissive enzymes are obviously of greater interest. We will discuss the various ThDP-dependent enzymes, their substrate speci¢city, and thence their synthetic potential with examples. To our knowledge, this ¢eld has not been reviewed before, although earlier reports compared ThDP-dependent enzymes to highlight other features [7,9]. 2. Speci¢c enzymes 2.1. Transketolase In recent years, transketolase (EC 2.2.1.1) has attracted much interest as catalyst for chemoenzymatic reactions. The enzyme therefore has been puri¢ed from spinach, yeast or recombinant E. coli strains [19^27] and is now available in su¤cient amounts to perform preparative scale reactions [14^18]. The main reactions of transketolase are depicted in Fig. 1. In general, an active glycolaldehyde group (K,Ldihydroxyethyl group) is transferred from a ketose donor 2, 4, 6 to an K-hydroxyaldehyde acceptor 1, 3, 5. Although the usual donor/acceptor pairs stem from the pentose-phosphate pathway of sugar metabolism, a wide variety of non-phosphorylated 2hydroxyaldehydes 7 (Table 1) are accepted at reasonable rates [7,27] to yield ketoses with a 3S,4R-con¢guration 9. As only hydroxyaldehydes with the con¢guration 2R are accepted by transketolase, Lhydroxyaldehydes 10 can be obtained as `spin-o¡' by kinetic resolution with good yield [28^30]. Aldehydes without a hydroxyl group at C2 11 are also transformed by transketolase leading to the product 12 [31^34] (Table 1), albeit with a signi¢cantly lower rate than the hydroxylated acceptors [16]. In contrast to the transketolases from spinach and yeast [31] no conversion of aromatic aldehydes as, e.g., benzalde-
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Fig. 1. Physiological and non-physiological reactions catalyzed by transketolase. R1 and R2 denote variable residues.
hyde or hydroxybenzaldehydes was detected with E. coli transketolase [27,33]. However, no structures of the reaction products from conversions of aromatic and heterocyclic substrates with transketolase have been shown yet. An earlier report claimed that the only non-aldehyde substrate 4-chloronitrosobenzene 13 was also used as acceptor [35], but so far the structure of the postulated product 14 has not been shown either. The use of hydroxypyruvate 8 as a donor substrate is of synthetic signi¢cance as it allows a practically irreversible product formation with carbon dioxide leaving the assay. While the transketolases from spinach, yeast and E. coli use 8 as a substrate [36^39], it is not accepted by the transketolases obtained from rat liver or human leukocytes [40,41]. For synthetic purposes the E. coli transketolase has a certain advantage over the enzymes from spinach and yeast, because the conversion of 8 with a rate of 60 U/mg [27] is signi¢cantly higher than the rates of 2 U/mg and 9 U/mg for the spinach and yeast enzymes [20,31]. 13 C-Labeled 8, which was generated from serine by the action of D-amino acid oxidase, has been used to synthesize the labeled sugar [1,213 C2]xylulose with spinach transketolase [42].
Using transketolase valuable natural compounds were synthesized easily (Fig. 2), whereas the chemical synthesis of these chiral substances a¡orded multistep syntheses with complex techniques involving introduction and removal of protective groups. Starting from a racemic mixture of 15 the chiral product 16 was obtained from the conversion with yeast transketolase and used for the chemical synthesis of the beetle pheromone K-exo-brevicomin 17 [43]. The enantioselective reaction of transketolase was further used for the chemoenzymatic synthesis of the glycosidase inhibitors fagomine 20, and 1,4-dideoxy-1,4imino-D-arabinitol 25. Starting points for the syntheses were the racemic mixtures of 18 and 23, which were converted to the chiral products 19 and 24 [44,45]. A similar strategy to gain precursors for the synthesis of 20 and 25 was used by Hecquet and co-workers [46]. The precursors 22 were obtained from the transketolase conversion of the racemic substrates 21 in the protected dithiane form. In all described syntheses transketolase generated two asymmetric centers at carbons C3 and C4 and therefore the use of a chiral starting compound was avoided. In a combined in vitro reaction involving several
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Table 1 Substrate range of transketolases from di¡erent organisms Acceptor substrate R1 -CHOH-CHO H CH3 CH2 OH CH2 F CH2 N3 CH2 CN CH2 NO2 CH2 SH CH2 OCH3 CH2 SCH3 CH2 SCH2 CH3 CH2 CH3 CH2 CH2 OH CHOH-CH3 D-erythro-CHOHCH2 OH D-threo-CHOHCH2 OH CH2 CH2 CH3 CH2 CH2 CHO CH2 CH2 CH2 CHO COH(CH3 )2 C(CH3 )3 CH=CH2 CrCH CH2 CH=CH2 (S)-CHOH-CH=CH2 (R)-CHOH-CH=CH2 D-ribo-CHOH-CHOH-CH2 OH D-gluco-CHOHCHOHCHOHCH2 OH 23 D-gluco-CHOHCHOHCHOHCH2 OPO3 CH(S2 C3 H6 ) 21 CH2CH(S2 C3 H6 ) 21 C6 H 5 CH2 C6 H5 CH2 OCH2 C6 H5 R2 -CHO H CH3 CH2 OCH3 CH2 SCH3 CH(OCH3 )2 CH2 CH3 CH2 CH2 OH COCH3 CH2 CH2 SCH3 CH2 CH2 CH3 CH2 CHOHCH3 CH2 CHOHCH2 OH CH=CHCH3 CH2 C6 H5
Source organism E. coli
Yeast
Spinach
[18,27,32^34] [17] [16,27,34]
[20,30,31] [20,30,31] [20] [28,31] [44] [28,45]
[55]
[16] [16]
[16] [17,27,32,33] [32,33]
[16] [17] [17,34] [17] [17]
[28,29] [28,30,31] [30] [28,30] [30,43] [30] [20,31] [31] [30] [31]
[16,27] [32,33] [16]
[59] [59] [34]
[28]
[17] [27]
[31]
[32,33] [34]
[56]
[30] [30] [30] [30] [30] [31] [20] [20]
[16,32^34] [16] [32,33]
[55]
[55] [31]
[31] [31] [31]
[57] *[58]
[46] [46]
[31,55] [55]
[55]
Substrate range of transketolase. (See Wood [54] for a compilation of transketolase substrates published before 1987; *[58], transketolase from B. subtilis.) K-Hydroxyaldehydes (R1 -CHOH-CO) as acceptor substrates with various residues R1 are given in the upper panel; aldehydes without an K-hydroxy group (R2 -CHO) and varying residues R2 are given in the lower panel.
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Fig. 2. Transketolase as catalyst in the chemoenzymatic synthesis of the pheromone K-exo-brevicomin (17) and of the glycosidase inhibitors fagomine (20) and 1,4-dideoxy-1,4-imino-D-arabinitol (25) [43^46]. HP, hydroxypyruvate as donor of C2 unit.
enzymes, a potential inhibitor of plant aromatic amino acid biosynthesis was prepared, 3-deoxy-D-arabinoheptulosonic acid 7-phosphonate which is derived from the natural compound, 3-deoxy-D-arabinoheptulosonic acid 7-phosphate 29 (DAHP; Fig. 3). The inhibitor was constructed by a combination of enzymatic steps including transketolase acting on fructose-6-P 6 with 1 as the C2 donor delivering 4 and erythrose-4-P 5 for the condensation with PEP 27 to form DAHP 29. 6 was generated in situ from 26 with hexokinase and the consumed ATP was regenerated with pyruvate kinase whereby 27 was converted to 28. The isolated DAHP 29 was then further reacted
Fig. 3. Generation of DAHP (29) with a multi-enzyme strategy using transketolase (TKT), hexokinase, pyruvate kinase, and DAHP synthase [47] starting from fructose (26) and ribose 5phosphate (1).
by chemical steps to yield the phosphonate. The yield of this chemoenzymatic approach was an order of magnitude better than the classical chemical path to the phosphonate [47]. Overexpression of transketolase was also used in vivo for an enhanced whole cell synthesis of DHAP 29, the ¢rst intermediate of the pathway leading to aromatic compounds [24,25]. Transketolase generates the same stereochemistry in its reaction products as fructose-1,6-diphosphate aldolase (EC 4.1.2.13). The commercially available enzyme from rabbit muscle (RAMA) has a broad substrate spectrum [1^8] and has been used for the chemoenzymatic synthesis of a variety of natural compounds [42,48^51]. However, Kobori and coworkers [30] pointed out that transketolase displays some advantages when compared with RAMA. Only transketolase can resolve racemic aldehyde substrates, so (less expensive) racemic mixtures can be used as starting materials for the synthesis of enantiomerically pure products. Furthermore the unphosphorylated sugar compounds, desired in most cases, are formed directly with transketolase whereas RAMA utilizes DHAP 33 and hence a second enzymatic step, dephosporylation with a phosphatase, is necessary to obtain the desired product. Additionally, some products can only be obtained with transketolase (Fig. 4). 31 is generated by transketolase from 30, whereas 32 does not serve as a substrate for the RAMA-catalyzed condensation with 33. The compounds 35 and 37 are synthesized with
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Fig. 4. The catalytic potential of transketolase in comparison to fructose-1,6-diphosphate aldolase from rabbit muscle (RAMA) [30]. HP, hydroxypyruvate.
transketolase from the aldehydes 34 and 36 which can easily be synthesized individually by chemical means [30]. In contrast, the RAMA catalyzed conversion of 30 followed by phosphatase treatment would lead to a mixture of the diastereomers 35 and 37. Last but not least, transketolase from E. coli [27] is more stable than the commercially available RAMA, which looses its activity completely within 2 days [52]. Though more stable bacterial fructose-1,6-diphosphate aldolases have been characterized meanwhile [49,53], transketolase can certainly
serve as an appropriate alternative to the use of aldolase as catalyst in chemoenzymatic syntheses. 2.2. Dihydroxyacetone synthase (formaldehyde transketolase) Dihydroxyacetone synthase (DHAS) occurs naturally in the peroxisomes of methylotrophic yeast [60] and is part of the xylulose monophosphate (XuMP) cycle for the assimilation of formaldehyde, the ¢rst metabolite of methanol oxidation [61,62]. DHAS cat-
Table 2 Substrate spectrum of DHAS from di¡erent organisms Substrate
Speci¢c activity (U/mg) Strain
Formaldehyde Acetaldehyde Propionaldehyde Butylaldehyde Heptylaldehyde Glycolaldehyde Glyceraldehyde Erythrose Ribose Erythrose 4-P Ribose 5-P Benzaldehyde Xylulose 5-P Hydroxypyruvate Ribulose 5-P
C. boidinii CBS5777
C. boidinii Kloeckera sp.
C. boidinii KD1
Acinetobacter sp. JC1 DSM 3803
0.8 0.35
3.9 3.9 5.5 5.8 4.6 4.0 3.7 3.2 0.9
5.7
0.66 0.1
8.3 5.2
1.2 0.5
3.2 0.4 3.9 2.2 0
2.9
1.1 0.5 0 1.0 0.55 0.8 0.3 0
5.2 2.5
Data from [66,69^72]. Omissions: no data available.
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1.3
0.44 0.18 0.2
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and adenylate kinase enabled the synthesis of 33, which can be used as a precursor for the generation of labeled sugars with DHAP-dependent aldolases [74]. 2.3. 1-Deoxyxylulose-5-phosphate synthase
Fig. 5. The use of dihydroxyacetone synthase in a multi-enzyme synthesis of 13 C-labeled dihydroxyacetone and its corresponding phosphate, starting from methanol [74]. DHAK, dihydroxyacetone kinase; DHAS, dihydroxyacetone synthase.
alyzes a transketolase analogue reaction with formaldehyde as acceptor and xylulose-5-phosphate as C2 donor [63]; therefore it is also referred to as formaldehyde transketolase as it shares features of a transketolase (size, sequence similarity [64], with formaldehyde as natural acceptor). The enzyme from di¡erent methylotrophic Candida boidinii strains has been characterized biochemically [65^71] and recently was detected in the methanol-utilizing carboxydo-bacterium Acinetobacter sp. DSM 3083 [72]. A recent article compared the bacterial DHAS with its counterparts in methylotrophic yeast C. boidinii (three di¡erent sources: [66,69^71]). All enzymes had in common to be rather unstable, and required Mg2 and ThDP. Donor substrates were xylulose 5phosphate in all cases, hydroxypyruvate (where tested), fructose 6-phosphate and, interestingly, also ribulose 5-phosphate for the bacterial enzyme (see Table 2; [72]). Formaldehyde was an acceptor in all cases, xylulose 5-P was the best donor. Other acceptors were glycolaldehyde, glyceraldehyde, acetaldehyde, ribose 5-P, and in some cases erythrose 4-P. Transketolase can compensate for the formaldehyde ¢xation in DHAS-negative mutants of the methylotrophic yeast Hansenula polymorpha [73]. DHAS has been used synthetically for the preparation of labeled [1,3-13 C]dihydroxyacetone 40 and [1,3-13 C]dihydroxyacetonephosphate 33 (Fig. 5) [74]. Using alcohol oxidase and catalase, 13 C-labeled 38 was transformed to formaldehyde 39 and irreversibly converted by DHAS to 40 with hydroxypyruvate 8 as C2 donor through the release of CO2 . A coupled reaction with dihydroxyacetone kinase (DHAK)
In a number of organisms, 1-deoxyxylulose-5phosphate (DXP) 41 has been identi¢ed with 13 C incorporation experiments as the precursor of a non-mevalonate pathway leading to isopentenyl diphosphate 43 (Fig. 6) [75^80]. Additionally, it has been demonstrated that 41 is involved in the biosynthesis of thiamin diphosphate 42 and pyridoxal phosphate 44 in bacteria [81^85]. Formation of 1-deoxyD-threo-pentulose has recently been reinvestigated in the authors' laboratory. We detected a novel enzyme in E. coli (1-deoxyxylulose-5-phosphate synthase) which is ThDP-dependent and catalyzes the formation of 1-deoxyxylulose from pyruvate and glyceraldehyde, or of DXP from pyruvate and glyceraldehyde-3-phosphate [86]. No other substrates have been tested so far. The enzyme is related to transketolases and the E1 subunit of the pyruvate dehydrogenase complex but belongs to a novel group of carboligases. The enzyme was successfully utilized for the enzymatic synthesis of 700 mg 41 (in its barium salt form) in a one-pot multi-enzyme synthesis (Fig. 7) [87]. Starting point was the RAMA catalyzed cleavage of 45 to yield 33 and 3 in equal amounts. 3 was converted irreversibly to 41 and CO2 by 1-deoxyxylulose-5-phosphate synthase and the use of 28 as the donor substrate. An almost complete conversion of 45 to 41 was achieved with triosephosphate iso-
Fig. 6. 1-Deoxyxylulose-5-phosphate (41) as precursor of three biosynthetic pathways eventually leading to thiamine diphosphate (42), isopentenyl diphosphate (43), and pyridoxal phosphate (44) [86].
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Fig. 7. The synthesis of 1-deoxyxylulose-5-phosphate (41) starting from fructose-1,6-bisphosphate (45) and pyruvate (28) with a multi-enzyme system containing rabbit muscle aldolase (RAMA), triosephosphate isomerase (TPI) and deoxyxylulose-5phosphate (DXP) synthase [86,87].
merase (TPI), which converted 33 and 3 reversibly. NMR analysis of the product con¢rmed that the Dthreo-pentulose was formed with a high enantiomeric excess. Since the understanding of the pathways leading to 42, 43 and 44 are at an early stage, large amounts of DXP are needed. Compared to the rather complicated chemical synthesis of the chiral compounds 1-deoxyxylulose and DXP [87^89] the enzymatic route to 1-deoxyxylulose and DXP certainly is advantageous. With the use of labeled pyruvate, the large scale enzymatic synthesis of labeled DXP for incorporation experiments should be possible. 2.4. Pyruvate dehydrogenase and other dehydrogenase complexes Pyruvate 28, acetoin 46 and 2-oxoglutarate 47 (Fig. 8) are the physiological donor substrates for the pyruvate dehydrogenase (PDH), acetoin dehydrogenase (AcoDH) and oxoglutarate dehydrogenase (OxoDH) complexes. The substrates are bound to the ThDP-containing E1 subunit 48, whereby CO2 is cleaved o¡ in the PDH and OxoDH complexes, and acetaldehyde is cleaved o¡ in the AcoDH catalyzed reaction. The intermediates are then transferred from the ThDP to the lipoamide moiety of the E2 subunits 49. In the view of chemoenzymatic syntheses, the transfer of the ThDP-bound intermediates to an acceptor aldehyde, leading to 50, is of potential interest and has been shown for all three complexes.
Pyruvate dehydrogenase in yeasts and animal tissues can convert the two-carbon unit of free acetaldehyde ^ by condensation with pyruvate ^ to acetoin; in higher animals the head-to-head combination of two acetaldehyde molecules has also been shown [90]. Puri¢ed PDH complex (PDHC) from pigeon breast muscle performed the stoichiometric condensation of two acetaldehyde molecules to acetoin without formation of by-products [90]. The acetoin formation was performed at 85% relative activity. The reaction required the presence of ThDP and Mg2 , and was essentially irreversible. Whereas acetoin is not a donor substrate for the PDH E1 subunit, 2,3-butanedione served as an alternative donor releasing acetic acid in the overall reaction of the PDH complex [91]. The enzymatic condensation of K-keto acids with various aldehydes is well established mainly through the work of Westerfeld and co-workers [92]. K-Oxoglutarate condenses with acetaldehyde in the presence of the K-oxoglutarate complex to form 5-hydroxy-4-ketohexanoic acid [93]. When condensation is with glyoxylate, two products are formed. The major product is 2-hydroxy-3-ketoadipic acid, which decarboxylates non-enzymatically to yield 5-hydroxy-4-ketovaleric acid [94]. The second, minor, product was identi¢ed as 2,3-dihydroxy-4-ketopimelic acid [95]. Pyruvate undergoes similar carboligase condensation reactions. The reaction with acetaldehyde has been mentioned earlier. Pyruvate reacts also with succinic semialdehyde to form 5keto-4-hydroxyhexanoic acid [96]. Kubasik and coworkers [92] showed the condensation reaction between pyruvate and glyoxylate to form acetol as
Fig. 8. Physiological and non-physiological reactions catalyzed by the E1 subunits of the acetoin dehydrogenase (AcoDH), pyruvate dehydrogenase (PDH), and oxoacid dehydrogenase (OxoDH) complexes.
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the major product and 1,2-dihydroxy-4-ketovaleric acid as a minor product with the E1 component of PDHC of E. coli being responsible for the condensation reaction between pyruvate and glyoxylate. The E. coli K-oxoglutarate complex was also shown to condense K-oxoglutarate and glyoxylate [97]. Carboligase reactions (acyloin-type condensation reactions) resulting in the formation of 1-deoxyketoses have been shown with cell-free extracts from various microorganisms [98]. Three types of reactions were shown for Bacillus subtilis, i.e. (I) pyruvate+aldoseCCO2 +1-deoxyketose (II) acetoin+aldoseCacetaldehyde+1-deoxyketose (III) methylacetoin+aldoseCacetone+1-deoxyketose Puri¢ed pyruvate dehydrogenase (EC 1.2.4.1) both from B. subtilis and E. coli (or bovine heart PDC) was shown to perform reaction I while reactions II and III were attributed to partially puri¢ed acetoin dehydrogenase (AccDh). Both enzymes required ThDP and Mg2 as cofactors. Among the aldoses which were used as acceptor compounds were glycolaldehyde, D- and L-glyceraldehyde, D-erythrose and D-threose. Products were 1-deoxyerythrulose, 1-deoxy-D-threo-pentulose, 1-deoxy-L-threo-pentulose, 1deoxy-D-fructose, and 1-deoxy-D-sorbose plus 1-deoxy-D-tagatose when D-threose was the acceptor [99,100]. For a further discussion, see Section 2. 2.5. Pyruvate decarboxylase Pyruvate decarboxylase (PDC; EC 4.1.1.1), a widely distributed enzyme playing a role in glycolysis and ethanol fermentations, has been puri¢ed from yeast [101,102], plants [103,104], and some bacteria as the ethanol producing Zymomonas mobilis [105,106]. Well known are side activities of PDC as carboligase (for details see chapter of M. Pohl in this issue) where the active acetaldehyde which is bound to the coenzyme is condensed with a second aldehyde as a co-substrate [107,108]. The condensation of benzaldehyde 52 with pyruvate 28 as the donor substrate yields (R)-1-hydroxy1-phenylpropan-2-one (phenylacetylcarbinol) 51 (Fig. 9) [109], the key intermediate in the synthesis of (3)-ephedrine 53. This biotransformation is exploited industrially since 1932 using whole yeast cells [11]. The reaction of 52 and 28 has also been studied extensively using, e.g., partially puri¢ed PDC or the
237
Fig. 9. Stereochemistry of the carboligase reactions with aromatic and aliphatic aldehydes catalyzed by pyruvate decarboxylase (PDC) from yeast and Z. mobilis, and the conversion of aromatic compounds by benzoylformate decarboxylase (BFD).
immobilized enzyme from C. utilis in enzyme reactors [110^113]. Other carboligase reactions of PDC are the formation of acetoin and chiral K-hydroxyketones [10]. A certain drawback is the low condensation activity on benzaldehyde of wild-type PDC from Z. mobilis [12,114]; however, the ratio of carboligase activity to decarboxylase activity could be improved recently by site-directed mutagenesis of the PDC [115]. Interestingly, acetoin and lactaldehyde synthesized from acetaldehyde 56 and pyruvate or glyoxylate 55 as alternative donors by PDC from yeast and Z. mobilis or wheat germ showed opposite con¢gurations [107,108,116]. The R-con¢gurated acyloin 54 was generated with the yeast enzyme in moderate enantiomeric excesses while the catalysis with the enzymes from Z. mobilis and from wheat germ led to the product with the S-con¢guration 57. PDC from Z. mobilis showed carboligase activity with propanal, heterocyclic aldehydes as furaldehydes or thiophene-3-aldehyde, and with halogenated aromatic aldehydes (£uoro or chlorophenyl) [117]. 2.6. Benzoylformate decarboxylase Benzoylformate decarboxylase (benzoylformate carboxylyase; BFD; EC 4.1.1.7) is a rare enzyme which catalyzes the formation of benzaldehyde 52 from benzoylformate 58 (Fig. 9), an K-keto acid, in
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Fig. 10. Physiological reactions catalyzed by the ThDP-dependent enzymes phosphoketolase, indolepyruvate decarboxylase (IPDC), acetohydroxyacid synthase (AHAS), acetolactate synthase (ALS), glyoxylate carboligase (GLC), and SHCHC synthase.
the catabolism of aromatic compounds by Pseudomonas putida. The enzyme has been puri¢ed and characterized from this organism [118,119], or from Acinetobacter calcoaceticus [120], and recently the crystallization and structure determination have been reported [121,122]. The proposed reaction mechanism is analogous to the formation of acetaldehyde by PDC. Only benzoylformate or para-substituted benzoylformates (methylbenzoylformate, £uoromethylbenzoylformate, hydroxymethylbenzoylformate) have been shown as substrates, whereas
pyruvate, K-ketobutyrate or K-ketoglutarate are not substrates [118]. BFD, in a side reaction with an excess of acetaldehyde 56, performs also an acyloin formation by condensing benzoylformate 58 and acetaldehyde 56 to (S)-2-hydroxypropiophenone 59 with an enantiomeric excess of 91^92% [119,120,123,124]. PDC from yeast, in contrast, performs an acyloin condensation which leads to the product 51 with the opposite chirality and with an exchange of the positions of the keto and hydroxyl groups at C2 and C3.
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2.7. Miscellaneous ThDP enzymes Phosphoketolases (EC 4.1.2.22.) are found in some lactic acid bacteria (Bi¢dobacterium, Leuconostoc sp., Lactobacillus plantarum) and other bacteria (Acetobacter xylinum) and catalyze an irreversible ThDPdependent phosphorolytic reaction splitting fructose 6-phosphate 6 (or xylulose 5-P 2) plus inorganic phosphate to yield erythrose 4-P 5 (glyceraldehyde 3-P 3) and acetylphosphate 60 (Fig. 10). They are landmark enzymes of so-called fructose-6-phosphate shunts. Besides there sugar-splitting capabilities, little is known about side reactions, especially their synthetic potential has not been evaluated further. Phosphoketolase from Leuconostoc mesenteroides was found to accept fructose 6-P, xylulose 5-P, hydroxypyruvate, and glycolaldehyde as substrates [125], and arsenate replaced phosphate so that acetate was the end product. In the presence of ferricyanide as an electron acceptor, PK from L. plantarum or Leu. mesenteroides formed glycolic acid instead of acetylP from xylulose 5-P, fructose 6-P, or hydroxypyruvate [126,127]. Km values for fructose 6-P from several Bi¢dobacteria were determined in the range from 11.5 to 26 mM [128] compared with 2.5 mM for the A. xylinum enzyme [129]; for the L. mesenteroides enzyme the Km value for fructose 6-P was 29 mM, and 4.7 mM for xylulose 5-P. The enzyme has been puri¢ed from several organisms [125,128,130], but no gene has yet been cloned or its sequence been reported so far. Indolepyruvate decarboxylase (IPDC; EC 4.1.1.74) catalyzes the decarboxylation of indolepyruvate 61 (derived from L-tryptophan) to indole-3-acetaldehyde 62 and is thus involved in the biosynthesis of this latter plant hormone compound. The enzyme from the plant-associated bacterium Enterobacter cloacae has been puri¢ed and characterized from recombinant E. coli cells [131^133]. Besides indole-3-pyruvic acid, IPDC also decarboxylated pyruvic acid (19% relative activity), but was inactive with indole-3-lactic acid, oxaloacetic acid, a.o. organic acids. Thus, IPDC is highly speci¢c and has so far not been used for synthetic purposes. The enzyme has, to our knowledge, not been investigated for any carboligase activity with aldehydes as acceptor substrates yet.
239
Bacteria contain several isozymes of acetolactate synthases (ALS, EC 4.1.3.18), also known as acetohydroxyacid synthases (AHAS), which are ThDP-dependent and catalyze the condensation of pyruvate 63 (R1 = H) either with another pyruvate molecule 63 (R2 = H), or with K-ketobutyrate 64 (R2 = CH3 ) to form K-acetolactate 64 (R1 = R2 = H) or K-aceto-Khydroxybutyrate 64 (R1 = H, R2 = CH3 ), respectively; ALS play important roles in the biosynthesis of the amino acids valine, leucine, isoleucine, and of the vitamin pantothenate [134,135]. ALS II from Salmonella typhimurium, besides the two reactions shown above, K-ketobutyrate 63 (R1 = R2 = CH3 ) is condensed with itself to form K-propio-K-hydroxybutyrate 64 (R1 = R2 = CH3 ) and CO2 (at about 20% the rate of the homologous condensation of pyruvate with a Km for ketobutyrate of about 10 mM) [136]. ALS III of E. coli did not carry out this non-physiological homologous condensation [134]. Glyoxylate carboligase (GCL; EC 4.1.1.47) from E. coli is known to catalyze the condensation of two glyoxylate molecules 65 to form tartronate semialdehyde 66 plus CO2 [137]. GCL is part of a pathway in the utilization of glycolate or glyoxylate in E. coli. The gene has been cloned and the enzyme characterized [138]. GCL reaction is analogous to that of AHAS isozymes and GCL belongs to a protein family which consists of AHAS and pyruvate oxidases with the common feature that all enzymes contain FAD as additional cofactor. Like pyruvate oxidase (POX; EC 1.2.3.3) [139], GCL was able to catalyze the AHAS reaction at a very low rate, e.g. about 1034 of that of pyruvate oxidase activity, and 3.8U1034 of GCL activity, respectively; AHAS III in turn was able to perform the GCL activity at 3.6U1032 [138]. In E. coli and in other bacteria, menaquinone is formed via a route with o-succinylbenzoic acid (OSB) as intermediate. OSB is formed by dehydration from 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC). SHCHC 68 is formed from isochorismate 67 and K-ketoglutarate 47, with subsequent loss of pyruvate 28 and CO2 , [140^143] by the gene product of menD, a bifunctional ThDP-dependent SHCHC synthase/K-oxoglutarate decarboxylase [144]. This K-oxoglutarate decarboxylase can be clearly distinguished from the usual E1 of the Oxo-
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DH complex (EC 1.2.4.2) [142,145,146]. No side reactions of this enzyme on other compounds have been reported yet. 3. Conclusions and outlook Only the ThDP-dependent enzymes transketolase, pyruvate decarboxylase and, partially, benzoylformate decarboxylase have been exploited for chemoenzymatic syntheses so far. In recent years, most of the ThDP-dependent enzymes known so far have been cloned (with the only exception phosphoketolase) and can be overproduced in recombinant strains. Thus, with a good availability of most of the enzymes, a closer look towards the synthetic potential of these enzymes will become an easy and rewarding task for chemists and biotechnologists. Another approach to make ThDP-dependent enzymes even more interesting for chemoenzymatic syntheses could be the broadening of the substrate spectra with molecular engineering. The three-dimensional structures of several ThDP-dependent enzymes have become available in recent years. Structural data for PDC from Saccharomyces uvarum [147] and S. cerevisiae [148], transketolase from S. cerevisiae [149,150], and pyruvate oxidase from L. plantarum [151] are deposited in the Protein Data Bank. Based on the structural information, investigations in the reaction mechanism of ThDP-dependent enzymes were carried out successfully [152^155]. A better understanding of the substrate binding was obtained from 3D structures with analogues of reaction intermediates and bound substrate [156^ 158], as well as amino acid exchanges in the substrate channel of transketolase [16,17,158^160] as well as PDC [161,162]. The 3D structures were used for changing amino acids in the active center of the respective enzymes from di¡erent organisms [16,17,115,162] in order to change the enzymes' properties. A nice example is the replacement of Trp-392 by alanine in the PDC of Z. mobilis. This single mutation resulted in a reduced stability of the mutant enzyme, a 10-fold higher Km value for ThDP, and to a higher hydrophobicity in the active center of the mutant enzyme (as in the case of the yeast enzyme). This led to an improved carboligase reaction of the Trp-392 mutant enzyme of Z. mobilis with the
aromatic and bulky co-substrate benzaldehyde to yield phenylacetylcarbinol; the mutant produced four times more phenylacetylcarbinol in 1 h reaction than the wild-type enzyme [115] (see also above and paper by Iding et al. in this issue). Acknowledgements This work was funded by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich SFB380/B21. References [1] E.J. Toone, E.S. Simon, G.M. Whitesides, Tetrahedron Lett. 45 (1989) 5365^5421. [2] D.G. Drueckhammer, W.J. Hennen, R.L. Pederson, C.F. Barbas, C.M. Gautheron, T. Krach, C.H. Wong, Synthesis (1991) 499^525. [3] K. Faber, in: Biotransformations in Organic Chemistry, Springer Verlag, Berlin, 1992, pp. 204^220. [4] C.H. Wong, G.M. Whitesides, in: Enzymes in Synthetic Organic Chemistry, Pergamon, Oxford, 1994, pp. 195^251. [5] C.H. Wong, R.L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew. Chem. 107 (1995) 453^474. [6] H.J.M. Gijsen, L. Qiao, W. Fitz, C.H. Wong, Chem. Rev. 96 (1996) 443^473. [7] W.-D. Fessner, C. Walter, Top. Curr. Chem. 184 (1996) 97^ 194. [8] C.H. Wong, Annu. Rev. Microbiol. 51 (1997) 285^310. [9] L.O. Krampitz, Annu. Rev. Biochem. 38 (1969) 213^240. [10] D.H.G. Crout, S. Davies, R.J. Heath, C.O. Miles, R. Rathbone, B.E.P. Swoboda, M.B. Gravestone, Biocatalysis 9 (1994) 1^30. [11] German patent 548 459/1932, Verfahren zur Herstellung von 1-phenyl-2-methylaminopropan-1-ol, 1932. [12] S. Bringer-Meyer, H. Sahm, Biocatalysis 1 (1988) 321^331. [13] D.H.G. Crout, H. Dalton, D.W. Hutchinson, M. Miyagoshi, J. Chem. Soc. Perkin Trans. 1 (1991) 1329^1332. [14] M.D. Lilly, R. Chauhan, C. French, M. Gyamerah, G.R. Hobbs, A. Humphrey, M. Isupov, J.A. Littlechild, R.K. Mitra, K.G. Morris, M. Rupprecht, N.J. Turner, J.M. Ward, A.J. Willets, J.M. Woodley, Ann. NY Acad. Sci. 782 (1996) 513^525. [15] G.R. Hobbs, R.K. Mitra, R.P. Chauhan, J.M. Woodley, M.D. Lilly, J. Biotechnol. 45 (1996) 173^179. [16] U. Scho«rken, H. Sahm, G.A. Sprenger, in: H. Bisswanger, A. Schellenberger (Eds.), Biochemistry and Physiology of Thiamin Diphosphate Enzymes, A and C Intemann Verlag, Prien, 1996, pp. 543^553. [17] U. Scho«rken, Ph.D. thesis, Ju«l. Ber. No. 3418, University of Du«sseldorf, 1997.
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BBAPRO 35653 26-6-98
Review
Thiamin-dependent enzymes as catalysts in chemoenzymatic syntheses Ulrich Scho«rken, Georg A. Sprenger * Institut fu«r Biotechnologie 1, Forschungszentrum Ju«lich GmbH, P.O. Box 1913, D-52425 Ju«lich, Germany Received 18 November 1997; accepted 12 February 1998
Abstract Enzymes are increasingly being used to perform regio- and enantioselective reactions in chemoenzymatic syntheses. To utilize enzymes for unphysiological reactions and to yield novel products, a broad substrate spectrum is desirable. Thiamin diphosphate (ThDP)-dependent enzymes vary in their substrate tolerance from rather strict substrate specificity (phosphoketolases, glyoxylate carboligase) to more permissive enzymes (transketolase, dihydroxyacetone synthase, pyruvate decarboxylase) and therefore differ in their potential to be used as biocatalysts. We give an overview of the known substrate spectra of ThDP-dependent enzymes and present examples of multi-enzyme or chemoenzymatic approaches which involve ThDP-dependent enzymes as biocatalysts to obtain pharmaceutical compounds as ephedrine and glycosidase inhibitors, sex pheromones as exo-brevicomin, 13 C-labeled metabolites, and other intermediates as 1-deoxyxylulose 5-phosphate, a precursor of vitamins and isoprenoids. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Thiamin diphosphate; Transketolase; Pyruvate dehydrogenase; Pyruvate decarboxylase; 1-Deoxyxylulose 5-phosphate
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
230
2.
Speci¢c enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Transketolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Dihydroxyacetone synthase (formaldehyde transketolase) . . . 2.3. 1-Deoxyxylulose-5-phosphate synthase . . . . . . . . . . . . . . . . . 2.4. Pyruvate dehydrogenase and other dehydrogenase complexes 2.5. Pyruvate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Benzoylformate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . 2.7. Miscellaneous ThDP enzymes . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . .
230 230 234 235 236 237 237 239
3.
Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
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* Corresponding author. Fax: +49 (2461) 612710; E-mail: [email protected] 0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 0 7 1 - 5
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U. Scho«rken, G.A. Sprenger / Biochimica et Biophysica Acta 1385 (1998) 229^243
1. Introduction Enzymes are ¢nding increasing acceptance as catalysts in pure and applied chemistry especially for their intrinsic chiral function as they deliver a certain enantiomer at a high enantiomer excess (% ee), combined with a high catalytic e¤ciency. In view of an increasing demand for enantiomerically pure compounds for, e.g., pharmaceutical products, the bene¢ts of enzymes are obvious. Environmental issues are also in favor of enzymes as enzymes help to save on hazardous compounds, organic solvents and other chemical waste. Especially in the ¢eld of carbohydrate chemistry, the inherent multifunctionality of sugars is an enormous task for an organic chemist who has to use a plethora of protective groups in order to prevent unwanted reactions of the hydroxyl, keto, or phosphate groups. A variety of enzymes, mostly lyases and aldolases, have been used so far to synthesize complex sugars, sugar analogues and other biologically important natural compounds [1^ 8]. Thiamin-dependent enzymes have been used for quite a while as catalysts in chemoenzymatic syntheses [3,7^10]. As far as we can tell, the earliest commercial utilization of a thiamin-dependent enzyme, and one of the ¢rst biotransformation processes to be commercialized at all, is the production with whole yeast cells of phenylacetylcarbinol, a precursor of L-ephedrine ([11], Knoll procedure), a process which is in use since the 1930s. It has been established meanwhile [12,13] that the underlying principle of reaction (benzaldehyde plus pyruvate to phenylacetylcarbinol) is being catalyzed by pyruvate decarboxylase. An in-depth treatise of this case is given in this issue by Iding et al. Nowadays, the enzyme which has also attracted a lot of interest in chemoenzymatic syntheses is transketolase from various microorganisms or from plants (e.g. spinach) as it has been used for various carboncarbon (C-C) bonding reactions. A certain drawback, however, has been the availability of commercial enzymes at a good quality, reasonable prices, and a reliable supply from biological sources. With the advent of molecular biology and the use of highly productive recombinant microorganisms, these issues are of lesser importance as it has been shown that enzymes as transketolase from Escherichia coli can be
supplied in amounts surpassing 1 million units easily [14^18]. This should help to increase the acceptance of enzymes for chemoenzymatic syntheses. In most ThDP-dependent reactions the holoenzyme catalyzes a carbon-to-carbon-bond cleavage of the substrate which is followed by a condensation of a portion of the cleaved substrate (associated with ThDP) with an acceptor to form the product. The nature of the acceptor determines whether the enzyme is speci¢c in its reaction or more permissive. For synthetic purposes permissive enzymes are obviously of greater interest. We will discuss the various ThDP-dependent enzymes, their substrate speci¢city, and thence their synthetic potential with examples. To our knowledge, this ¢eld has not been reviewed before, although earlier reports compared ThDP-dependent enzymes to highlight other features [7,9]. 2. Speci¢c enzymes 2.1. Transketolase In recent years, transketolase (EC 2.2.1.1) has attracted much interest as catalyst for chemoenzymatic reactions. The enzyme therefore has been puri¢ed from spinach, yeast or recombinant E. coli strains [19^27] and is now available in su¤cient amounts to perform preparative scale reactions [14^18]. The main reactions of transketolase are depicted in Fig. 1. In general, an active glycolaldehyde group (K,Ldihydroxyethyl group) is transferred from a ketose donor 2, 4, 6 to an K-hydroxyaldehyde acceptor 1, 3, 5. Although the usual donor/acceptor pairs stem from the pentose-phosphate pathway of sugar metabolism, a wide variety of non-phosphorylated 2hydroxyaldehydes 7 (Table 1) are accepted at reasonable rates [7,27] to yield ketoses with a 3S,4R-con¢guration 9. As only hydroxyaldehydes with the con¢guration 2R are accepted by transketolase, Lhydroxyaldehydes 10 can be obtained as `spin-o¡' by kinetic resolution with good yield [28^30]. Aldehydes without a hydroxyl group at C2 11 are also transformed by transketolase leading to the product 12 [31^34] (Table 1), albeit with a signi¢cantly lower rate than the hydroxylated acceptors [16]. In contrast to the transketolases from spinach and yeast [31] no conversion of aromatic aldehydes as, e.g., benzalde-
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231
Fig. 1. Physiological and non-physiological reactions catalyzed by transketolase. R1 and R2 denote variable residues.
hyde or hydroxybenzaldehydes was detected with E. coli transketolase [27,33]. However, no structures of the reaction products from conversions of aromatic and heterocyclic substrates with transketolase have been shown yet. An earlier report claimed that the only non-aldehyde substrate 4-chloronitrosobenzene 13 was also used as acceptor [35], but so far the structure of the postulated product 14 has not been shown either. The use of hydroxypyruvate 8 as a donor substrate is of synthetic signi¢cance as it allows a practically irreversible product formation with carbon dioxide leaving the assay. While the transketolases from spinach, yeast and E. coli use 8 as a substrate [36^39], it is not accepted by the transketolases obtained from rat liver or human leukocytes [40,41]. For synthetic purposes the E. coli transketolase has a certain advantage over the enzymes from spinach and yeast, because the conversion of 8 with a rate of 60 U/mg [27] is signi¢cantly higher than the rates of 2 U/mg and 9 U/mg for the spinach and yeast enzymes [20,31]. 13 C-Labeled 8, which was generated from serine by the action of D-amino acid oxidase, has been used to synthesize the labeled sugar [1,213 C2]xylulose with spinach transketolase [42].
Using transketolase valuable natural compounds were synthesized easily (Fig. 2), whereas the chemical synthesis of these chiral substances a¡orded multistep syntheses with complex techniques involving introduction and removal of protective groups. Starting from a racemic mixture of 15 the chiral product 16 was obtained from the conversion with yeast transketolase and used for the chemical synthesis of the beetle pheromone K-exo-brevicomin 17 [43]. The enantioselective reaction of transketolase was further used for the chemoenzymatic synthesis of the glycosidase inhibitors fagomine 20, and 1,4-dideoxy-1,4imino-D-arabinitol 25. Starting points for the syntheses were the racemic mixtures of 18 and 23, which were converted to the chiral products 19 and 24 [44,45]. A similar strategy to gain precursors for the synthesis of 20 and 25 was used by Hecquet and co-workers [46]. The precursors 22 were obtained from the transketolase conversion of the racemic substrates 21 in the protected dithiane form. In all described syntheses transketolase generated two asymmetric centers at carbons C3 and C4 and therefore the use of a chiral starting compound was avoided. In a combined in vitro reaction involving several
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Table 1 Substrate range of transketolases from di¡erent organisms Acceptor substrate R1 -CHOH-CHO H CH3 CH2 OH CH2 F CH2 N3 CH2 CN CH2 NO2 CH2 SH CH2 OCH3 CH2 SCH3 CH2 SCH2 CH3 CH2 CH3 CH2 CH2 OH CHOH-CH3 D-erythro-CHOHCH2 OH D-threo-CHOHCH2 OH CH2 CH2 CH3 CH2 CH2 CHO CH2 CH2 CH2 CHO COH(CH3 )2 C(CH3 )3 CH=CH2 CrCH CH2 CH=CH2 (S)-CHOH-CH=CH2 (R)-CHOH-CH=CH2 D-ribo-CHOH-CHOH-CH2 OH D-gluco-CHOHCHOHCHOHCH2 OH 23 D-gluco-CHOHCHOHCHOHCH2 OPO3 CH(S2 C3 H6 ) 21 CH2CH(S2 C3 H6 ) 21 C6 H 5 CH2 C6 H5 CH2 OCH2 C6 H5 R2 -CHO H CH3 CH2 OCH3 CH2 SCH3 CH(OCH3 )2 CH2 CH3 CH2 CH2 OH COCH3 CH2 CH2 SCH3 CH2 CH2 CH3 CH2 CHOHCH3 CH2 CHOHCH2 OH CH=CHCH3 CH2 C6 H5
Source organism E. coli
Yeast
Spinach
[18,27,32^34] [17] [16,27,34]
[20,30,31] [20,30,31] [20] [28,31] [44] [28,45]
[55]
[16] [16]
[16] [17,27,32,33] [32,33]
[16] [17] [17,34] [17] [17]
[28,29] [28,30,31] [30] [28,30] [30,43] [30] [20,31] [31] [30] [31]
[16,27] [32,33] [16]
[59] [59] [34]
[28]
[17] [27]
[31]
[32,33] [34]
[56]
[30] [30] [30] [30] [30] [31] [20] [20]
[16,32^34] [16] [32,33]
[55]
[55] [31]
[31] [31] [31]
[57] *[58]
[46] [46]
[31,55] [55]
[55]
Substrate range of transketolase. (See Wood [54] for a compilation of transketolase substrates published before 1987; *[58], transketolase from B. subtilis.) K-Hydroxyaldehydes (R1 -CHOH-CO) as acceptor substrates with various residues R1 are given in the upper panel; aldehydes without an K-hydroxy group (R2 -CHO) and varying residues R2 are given in the lower panel.
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233
Fig. 2. Transketolase as catalyst in the chemoenzymatic synthesis of the pheromone K-exo-brevicomin (17) and of the glycosidase inhibitors fagomine (20) and 1,4-dideoxy-1,4-imino-D-arabinitol (25) [43^46]. HP, hydroxypyruvate as donor of C2 unit.
enzymes, a potential inhibitor of plant aromatic amino acid biosynthesis was prepared, 3-deoxy-D-arabinoheptulosonic acid 7-phosphonate which is derived from the natural compound, 3-deoxy-D-arabinoheptulosonic acid 7-phosphate 29 (DAHP; Fig. 3). The inhibitor was constructed by a combination of enzymatic steps including transketolase acting on fructose-6-P 6 with 1 as the C2 donor delivering 4 and erythrose-4-P 5 for the condensation with PEP 27 to form DAHP 29. 6 was generated in situ from 26 with hexokinase and the consumed ATP was regenerated with pyruvate kinase whereby 27 was converted to 28. The isolated DAHP 29 was then further reacted
Fig. 3. Generation of DAHP (29) with a multi-enzyme strategy using transketolase (TKT), hexokinase, pyruvate kinase, and DAHP synthase [47] starting from fructose (26) and ribose 5phosphate (1).
by chemical steps to yield the phosphonate. The yield of this chemoenzymatic approach was an order of magnitude better than the classical chemical path to the phosphonate [47]. Overexpression of transketolase was also used in vivo for an enhanced whole cell synthesis of DHAP 29, the ¢rst intermediate of the pathway leading to aromatic compounds [24,25]. Transketolase generates the same stereochemistry in its reaction products as fructose-1,6-diphosphate aldolase (EC 4.1.2.13). The commercially available enzyme from rabbit muscle (RAMA) has a broad substrate spectrum [1^8] and has been used for the chemoenzymatic synthesis of a variety of natural compounds [42,48^51]. However, Kobori and coworkers [30] pointed out that transketolase displays some advantages when compared with RAMA. Only transketolase can resolve racemic aldehyde substrates, so (less expensive) racemic mixtures can be used as starting materials for the synthesis of enantiomerically pure products. Furthermore the unphosphorylated sugar compounds, desired in most cases, are formed directly with transketolase whereas RAMA utilizes DHAP 33 and hence a second enzymatic step, dephosporylation with a phosphatase, is necessary to obtain the desired product. Additionally, some products can only be obtained with transketolase (Fig. 4). 31 is generated by transketolase from 30, whereas 32 does not serve as a substrate for the RAMA-catalyzed condensation with 33. The compounds 35 and 37 are synthesized with
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Fig. 4. The catalytic potential of transketolase in comparison to fructose-1,6-diphosphate aldolase from rabbit muscle (RAMA) [30]. HP, hydroxypyruvate.
transketolase from the aldehydes 34 and 36 which can easily be synthesized individually by chemical means [30]. In contrast, the RAMA catalyzed conversion of 30 followed by phosphatase treatment would lead to a mixture of the diastereomers 35 and 37. Last but not least, transketolase from E. coli [27] is more stable than the commercially available RAMA, which looses its activity completely within 2 days [52]. Though more stable bacterial fructose-1,6-diphosphate aldolases have been characterized meanwhile [49,53], transketolase can certainly
serve as an appropriate alternative to the use of aldolase as catalyst in chemoenzymatic syntheses. 2.2. Dihydroxyacetone synthase (formaldehyde transketolase) Dihydroxyacetone synthase (DHAS) occurs naturally in the peroxisomes of methylotrophic yeast [60] and is part of the xylulose monophosphate (XuMP) cycle for the assimilation of formaldehyde, the ¢rst metabolite of methanol oxidation [61,62]. DHAS cat-
Table 2 Substrate spectrum of DHAS from di¡erent organisms Substrate
Speci¢c activity (U/mg) Strain
Formaldehyde Acetaldehyde Propionaldehyde Butylaldehyde Heptylaldehyde Glycolaldehyde Glyceraldehyde Erythrose Ribose Erythrose 4-P Ribose 5-P Benzaldehyde Xylulose 5-P Hydroxypyruvate Ribulose 5-P
C. boidinii CBS5777
C. boidinii Kloeckera sp.
C. boidinii KD1
Acinetobacter sp. JC1 DSM 3803
0.8 0.35
3.9 3.9 5.5 5.8 4.6 4.0 3.7 3.2 0.9
5.7
0.66 0.1
8.3 5.2
1.2 0.5
3.2 0.4 3.9 2.2 0
2.9
1.1 0.5 0 1.0 0.55 0.8 0.3 0
5.2 2.5
Data from [66,69^72]. Omissions: no data available.
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1.3
0.44 0.18 0.2
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235
and adenylate kinase enabled the synthesis of 33, which can be used as a precursor for the generation of labeled sugars with DHAP-dependent aldolases [74]. 2.3. 1-Deoxyxylulose-5-phosphate synthase
Fig. 5. The use of dihydroxyacetone synthase in a multi-enzyme synthesis of 13 C-labeled dihydroxyacetone and its corresponding phosphate, starting from methanol [74]. DHAK, dihydroxyacetone kinase; DHAS, dihydroxyacetone synthase.
alyzes a transketolase analogue reaction with formaldehyde as acceptor and xylulose-5-phosphate as C2 donor [63]; therefore it is also referred to as formaldehyde transketolase as it shares features of a transketolase (size, sequence similarity [64], with formaldehyde as natural acceptor). The enzyme from di¡erent methylotrophic Candida boidinii strains has been characterized biochemically [65^71] and recently was detected in the methanol-utilizing carboxydo-bacterium Acinetobacter sp. DSM 3083 [72]. A recent article compared the bacterial DHAS with its counterparts in methylotrophic yeast C. boidinii (three di¡erent sources: [66,69^71]). All enzymes had in common to be rather unstable, and required Mg2 and ThDP. Donor substrates were xylulose 5phosphate in all cases, hydroxypyruvate (where tested), fructose 6-phosphate and, interestingly, also ribulose 5-phosphate for the bacterial enzyme (see Table 2; [72]). Formaldehyde was an acceptor in all cases, xylulose 5-P was the best donor. Other acceptors were glycolaldehyde, glyceraldehyde, acetaldehyde, ribose 5-P, and in some cases erythrose 4-P. Transketolase can compensate for the formaldehyde ¢xation in DHAS-negative mutants of the methylotrophic yeast Hansenula polymorpha [73]. DHAS has been used synthetically for the preparation of labeled [1,3-13 C]dihydroxyacetone 40 and [1,3-13 C]dihydroxyacetonephosphate 33 (Fig. 5) [74]. Using alcohol oxidase and catalase, 13 C-labeled 38 was transformed to formaldehyde 39 and irreversibly converted by DHAS to 40 with hydroxypyruvate 8 as C2 donor through the release of CO2 . A coupled reaction with dihydroxyacetone kinase (DHAK)
In a number of organisms, 1-deoxyxylulose-5phosphate (DXP) 41 has been identi¢ed with 13 C incorporation experiments as the precursor of a non-mevalonate pathway leading to isopentenyl diphosphate 43 (Fig. 6) [75^80]. Additionally, it has been demonstrated that 41 is involved in the biosynthesis of thiamin diphosphate 42 and pyridoxal phosphate 44 in bacteria [81^85]. Formation of 1-deoxyD-threo-pentulose has recently been reinvestigated in the authors' laboratory. We detected a novel enzyme in E. coli (1-deoxyxylulose-5-phosphate synthase) which is ThDP-dependent and catalyzes the formation of 1-deoxyxylulose from pyruvate and glyceraldehyde, or of DXP from pyruvate and glyceraldehyde-3-phosphate [86]. No other substrates have been tested so far. The enzyme is related to transketolases and the E1 subunit of the pyruvate dehydrogenase complex but belongs to a novel group of carboligases. The enzyme was successfully utilized for the enzymatic synthesis of 700 mg 41 (in its barium salt form) in a one-pot multi-enzyme synthesis (Fig. 7) [87]. Starting point was the RAMA catalyzed cleavage of 45 to yield 33 and 3 in equal amounts. 3 was converted irreversibly to 41 and CO2 by 1-deoxyxylulose-5-phosphate synthase and the use of 28 as the donor substrate. An almost complete conversion of 45 to 41 was achieved with triosephosphate iso-
Fig. 6. 1-Deoxyxylulose-5-phosphate (41) as precursor of three biosynthetic pathways eventually leading to thiamine diphosphate (42), isopentenyl diphosphate (43), and pyridoxal phosphate (44) [86].
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Fig. 7. The synthesis of 1-deoxyxylulose-5-phosphate (41) starting from fructose-1,6-bisphosphate (45) and pyruvate (28) with a multi-enzyme system containing rabbit muscle aldolase (RAMA), triosephosphate isomerase (TPI) and deoxyxylulose-5phosphate (DXP) synthase [86,87].
merase (TPI), which converted 33 and 3 reversibly. NMR analysis of the product con¢rmed that the Dthreo-pentulose was formed with a high enantiomeric excess. Since the understanding of the pathways leading to 42, 43 and 44 are at an early stage, large amounts of DXP are needed. Compared to the rather complicated chemical synthesis of the chiral compounds 1-deoxyxylulose and DXP [87^89] the enzymatic route to 1-deoxyxylulose and DXP certainly is advantageous. With the use of labeled pyruvate, the large scale enzymatic synthesis of labeled DXP for incorporation experiments should be possible. 2.4. Pyruvate dehydrogenase and other dehydrogenase complexes Pyruvate 28, acetoin 46 and 2-oxoglutarate 47 (Fig. 8) are the physiological donor substrates for the pyruvate dehydrogenase (PDH), acetoin dehydrogenase (AcoDH) and oxoglutarate dehydrogenase (OxoDH) complexes. The substrates are bound to the ThDP-containing E1 subunit 48, whereby CO2 is cleaved o¡ in the PDH and OxoDH complexes, and acetaldehyde is cleaved o¡ in the AcoDH catalyzed reaction. The intermediates are then transferred from the ThDP to the lipoamide moiety of the E2 subunits 49. In the view of chemoenzymatic syntheses, the transfer of the ThDP-bound intermediates to an acceptor aldehyde, leading to 50, is of potential interest and has been shown for all three complexes.
Pyruvate dehydrogenase in yeasts and animal tissues can convert the two-carbon unit of free acetaldehyde ^ by condensation with pyruvate ^ to acetoin; in higher animals the head-to-head combination of two acetaldehyde molecules has also been shown [90]. Puri¢ed PDH complex (PDHC) from pigeon breast muscle performed the stoichiometric condensation of two acetaldehyde molecules to acetoin without formation of by-products [90]. The acetoin formation was performed at 85% relative activity. The reaction required the presence of ThDP and Mg2 , and was essentially irreversible. Whereas acetoin is not a donor substrate for the PDH E1 subunit, 2,3-butanedione served as an alternative donor releasing acetic acid in the overall reaction of the PDH complex [91]. The enzymatic condensation of K-keto acids with various aldehydes is well established mainly through the work of Westerfeld and co-workers [92]. K-Oxoglutarate condenses with acetaldehyde in the presence of the K-oxoglutarate complex to form 5-hydroxy-4-ketohexanoic acid [93]. When condensation is with glyoxylate, two products are formed. The major product is 2-hydroxy-3-ketoadipic acid, which decarboxylates non-enzymatically to yield 5-hydroxy-4-ketovaleric acid [94]. The second, minor, product was identi¢ed as 2,3-dihydroxy-4-ketopimelic acid [95]. Pyruvate undergoes similar carboligase condensation reactions. The reaction with acetaldehyde has been mentioned earlier. Pyruvate reacts also with succinic semialdehyde to form 5keto-4-hydroxyhexanoic acid [96]. Kubasik and coworkers [92] showed the condensation reaction between pyruvate and glyoxylate to form acetol as
Fig. 8. Physiological and non-physiological reactions catalyzed by the E1 subunits of the acetoin dehydrogenase (AcoDH), pyruvate dehydrogenase (PDH), and oxoacid dehydrogenase (OxoDH) complexes.
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the major product and 1,2-dihydroxy-4-ketovaleric acid as a minor product with the E1 component of PDHC of E. coli being responsible for the condensation reaction between pyruvate and glyoxylate. The E. coli K-oxoglutarate complex was also shown to condense K-oxoglutarate and glyoxylate [97]. Carboligase reactions (acyloin-type condensation reactions) resulting in the formation of 1-deoxyketoses have been shown with cell-free extracts from various microorganisms [98]. Three types of reactions were shown for Bacillus subtilis, i.e. (I) pyruvate+aldoseCCO2 +1-deoxyketose (II) acetoin+aldoseCacetaldehyde+1-deoxyketose (III) methylacetoin+aldoseCacetone+1-deoxyketose Puri¢ed pyruvate dehydrogenase (EC 1.2.4.1) both from B. subtilis and E. coli (or bovine heart PDC) was shown to perform reaction I while reactions II and III were attributed to partially puri¢ed acetoin dehydrogenase (AccDh). Both enzymes required ThDP and Mg2 as cofactors. Among the aldoses which were used as acceptor compounds were glycolaldehyde, D- and L-glyceraldehyde, D-erythrose and D-threose. Products were 1-deoxyerythrulose, 1-deoxy-D-threo-pentulose, 1-deoxy-L-threo-pentulose, 1deoxy-D-fructose, and 1-deoxy-D-sorbose plus 1-deoxy-D-tagatose when D-threose was the acceptor [99,100]. For a further discussion, see Section 2. 2.5. Pyruvate decarboxylase Pyruvate decarboxylase (PDC; EC 4.1.1.1), a widely distributed enzyme playing a role in glycolysis and ethanol fermentations, has been puri¢ed from yeast [101,102], plants [103,104], and some bacteria as the ethanol producing Zymomonas mobilis [105,106]. Well known are side activities of PDC as carboligase (for details see chapter of M. Pohl in this issue) where the active acetaldehyde which is bound to the coenzyme is condensed with a second aldehyde as a co-substrate [107,108]. The condensation of benzaldehyde 52 with pyruvate 28 as the donor substrate yields (R)-1-hydroxy1-phenylpropan-2-one (phenylacetylcarbinol) 51 (Fig. 9) [109], the key intermediate in the synthesis of (3)-ephedrine 53. This biotransformation is exploited industrially since 1932 using whole yeast cells [11]. The reaction of 52 and 28 has also been studied extensively using, e.g., partially puri¢ed PDC or the
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Fig. 9. Stereochemistry of the carboligase reactions with aromatic and aliphatic aldehydes catalyzed by pyruvate decarboxylase (PDC) from yeast and Z. mobilis, and the conversion of aromatic compounds by benzoylformate decarboxylase (BFD).
immobilized enzyme from C. utilis in enzyme reactors [110^113]. Other carboligase reactions of PDC are the formation of acetoin and chiral K-hydroxyketones [10]. A certain drawback is the low condensation activity on benzaldehyde of wild-type PDC from Z. mobilis [12,114]; however, the ratio of carboligase activity to decarboxylase activity could be improved recently by site-directed mutagenesis of the PDC [115]. Interestingly, acetoin and lactaldehyde synthesized from acetaldehyde 56 and pyruvate or glyoxylate 55 as alternative donors by PDC from yeast and Z. mobilis or wheat germ showed opposite con¢gurations [107,108,116]. The R-con¢gurated acyloin 54 was generated with the yeast enzyme in moderate enantiomeric excesses while the catalysis with the enzymes from Z. mobilis and from wheat germ led to the product with the S-con¢guration 57. PDC from Z. mobilis showed carboligase activity with propanal, heterocyclic aldehydes as furaldehydes or thiophene-3-aldehyde, and with halogenated aromatic aldehydes (£uoro or chlorophenyl) [117]. 2.6. Benzoylformate decarboxylase Benzoylformate decarboxylase (benzoylformate carboxylyase; BFD; EC 4.1.1.7) is a rare enzyme which catalyzes the formation of benzaldehyde 52 from benzoylformate 58 (Fig. 9), an K-keto acid, in
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Fig. 10. Physiological reactions catalyzed by the ThDP-dependent enzymes phosphoketolase, indolepyruvate decarboxylase (IPDC), acetohydroxyacid synthase (AHAS), acetolactate synthase (ALS), glyoxylate carboligase (GLC), and SHCHC synthase.
the catabolism of aromatic compounds by Pseudomonas putida. The enzyme has been puri¢ed and characterized from this organism [118,119], or from Acinetobacter calcoaceticus [120], and recently the crystallization and structure determination have been reported [121,122]. The proposed reaction mechanism is analogous to the formation of acetaldehyde by PDC. Only benzoylformate or para-substituted benzoylformates (methylbenzoylformate, £uoromethylbenzoylformate, hydroxymethylbenzoylformate) have been shown as substrates, whereas
pyruvate, K-ketobutyrate or K-ketoglutarate are not substrates [118]. BFD, in a side reaction with an excess of acetaldehyde 56, performs also an acyloin formation by condensing benzoylformate 58 and acetaldehyde 56 to (S)-2-hydroxypropiophenone 59 with an enantiomeric excess of 91^92% [119,120,123,124]. PDC from yeast, in contrast, performs an acyloin condensation which leads to the product 51 with the opposite chirality and with an exchange of the positions of the keto and hydroxyl groups at C2 and C3.
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2.7. Miscellaneous ThDP enzymes Phosphoketolases (EC 4.1.2.22.) are found in some lactic acid bacteria (Bi¢dobacterium, Leuconostoc sp., Lactobacillus plantarum) and other bacteria (Acetobacter xylinum) and catalyze an irreversible ThDPdependent phosphorolytic reaction splitting fructose 6-phosphate 6 (or xylulose 5-P 2) plus inorganic phosphate to yield erythrose 4-P 5 (glyceraldehyde 3-P 3) and acetylphosphate 60 (Fig. 10). They are landmark enzymes of so-called fructose-6-phosphate shunts. Besides there sugar-splitting capabilities, little is known about side reactions, especially their synthetic potential has not been evaluated further. Phosphoketolase from Leuconostoc mesenteroides was found to accept fructose 6-P, xylulose 5-P, hydroxypyruvate, and glycolaldehyde as substrates [125], and arsenate replaced phosphate so that acetate was the end product. In the presence of ferricyanide as an electron acceptor, PK from L. plantarum or Leu. mesenteroides formed glycolic acid instead of acetylP from xylulose 5-P, fructose 6-P, or hydroxypyruvate [126,127]. Km values for fructose 6-P from several Bi¢dobacteria were determined in the range from 11.5 to 26 mM [128] compared with 2.5 mM for the A. xylinum enzyme [129]; for the L. mesenteroides enzyme the Km value for fructose 6-P was 29 mM, and 4.7 mM for xylulose 5-P. The enzyme has been puri¢ed from several organisms [125,128,130], but no gene has yet been cloned or its sequence been reported so far. Indolepyruvate decarboxylase (IPDC; EC 4.1.1.74) catalyzes the decarboxylation of indolepyruvate 61 (derived from L-tryptophan) to indole-3-acetaldehyde 62 and is thus involved in the biosynthesis of this latter plant hormone compound. The enzyme from the plant-associated bacterium Enterobacter cloacae has been puri¢ed and characterized from recombinant E. coli cells [131^133]. Besides indole-3-pyruvic acid, IPDC also decarboxylated pyruvic acid (19% relative activity), but was inactive with indole-3-lactic acid, oxaloacetic acid, a.o. organic acids. Thus, IPDC is highly speci¢c and has so far not been used for synthetic purposes. The enzyme has, to our knowledge, not been investigated for any carboligase activity with aldehydes as acceptor substrates yet.
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Bacteria contain several isozymes of acetolactate synthases (ALS, EC 4.1.3.18), also known as acetohydroxyacid synthases (AHAS), which are ThDP-dependent and catalyze the condensation of pyruvate 63 (R1 = H) either with another pyruvate molecule 63 (R2 = H), or with K-ketobutyrate 64 (R2 = CH3 ) to form K-acetolactate 64 (R1 = R2 = H) or K-aceto-Khydroxybutyrate 64 (R1 = H, R2 = CH3 ), respectively; ALS play important roles in the biosynthesis of the amino acids valine, leucine, isoleucine, and of the vitamin pantothenate [134,135]. ALS II from Salmonella typhimurium, besides the two reactions shown above, K-ketobutyrate 63 (R1 = R2 = CH3 ) is condensed with itself to form K-propio-K-hydroxybutyrate 64 (R1 = R2 = CH3 ) and CO2 (at about 20% the rate of the homologous condensation of pyruvate with a Km for ketobutyrate of about 10 mM) [136]. ALS III of E. coli did not carry out this non-physiological homologous condensation [134]. Glyoxylate carboligase (GCL; EC 4.1.1.47) from E. coli is known to catalyze the condensation of two glyoxylate molecules 65 to form tartronate semialdehyde 66 plus CO2 [137]. GCL is part of a pathway in the utilization of glycolate or glyoxylate in E. coli. The gene has been cloned and the enzyme characterized [138]. GCL reaction is analogous to that of AHAS isozymes and GCL belongs to a protein family which consists of AHAS and pyruvate oxidases with the common feature that all enzymes contain FAD as additional cofactor. Like pyruvate oxidase (POX; EC 1.2.3.3) [139], GCL was able to catalyze the AHAS reaction at a very low rate, e.g. about 1034 of that of pyruvate oxidase activity, and 3.8U1034 of GCL activity, respectively; AHAS III in turn was able to perform the GCL activity at 3.6U1032 [138]. In E. coli and in other bacteria, menaquinone is formed via a route with o-succinylbenzoic acid (OSB) as intermediate. OSB is formed by dehydration from 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC). SHCHC 68 is formed from isochorismate 67 and K-ketoglutarate 47, with subsequent loss of pyruvate 28 and CO2 , [140^143] by the gene product of menD, a bifunctional ThDP-dependent SHCHC synthase/K-oxoglutarate decarboxylase [144]. This K-oxoglutarate decarboxylase can be clearly distinguished from the usual E1 of the Oxo-
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DH complex (EC 1.2.4.2) [142,145,146]. No side reactions of this enzyme on other compounds have been reported yet. 3. Conclusions and outlook Only the ThDP-dependent enzymes transketolase, pyruvate decarboxylase and, partially, benzoylformate decarboxylase have been exploited for chemoenzymatic syntheses so far. In recent years, most of the ThDP-dependent enzymes known so far have been cloned (with the only exception phosphoketolase) and can be overproduced in recombinant strains. Thus, with a good availability of most of the enzymes, a closer look towards the synthetic potential of these enzymes will become an easy and rewarding task for chemists and biotechnologists. Another approach to make ThDP-dependent enzymes even more interesting for chemoenzymatic syntheses could be the broadening of the substrate spectra with molecular engineering. The three-dimensional structures of several ThDP-dependent enzymes have become available in recent years. Structural data for PDC from Saccharomyces uvarum [147] and S. cerevisiae [148], transketolase from S. cerevisiae [149,150], and pyruvate oxidase from L. plantarum [151] are deposited in the Protein Data Bank. Based on the structural information, investigations in the reaction mechanism of ThDP-dependent enzymes were carried out successfully [152^155]. A better understanding of the substrate binding was obtained from 3D structures with analogues of reaction intermediates and bound substrate [156^ 158], as well as amino acid exchanges in the substrate channel of transketolase [16,17,158^160] as well as PDC [161,162]. The 3D structures were used for changing amino acids in the active center of the respective enzymes from di¡erent organisms [16,17,115,162] in order to change the enzymes' properties. A nice example is the replacement of Trp-392 by alanine in the PDC of Z. mobilis. This single mutation resulted in a reduced stability of the mutant enzyme, a 10-fold higher Km value for ThDP, and to a higher hydrophobicity in the active center of the mutant enzyme (as in the case of the yeast enzyme). This led to an improved carboligase reaction of the Trp-392 mutant enzyme of Z. mobilis with the
aromatic and bulky co-substrate benzaldehyde to yield phenylacetylcarbinol; the mutant produced four times more phenylacetylcarbinol in 1 h reaction than the wild-type enzyme [115] (see also above and paper by Iding et al. in this issue). Acknowledgements This work was funded by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich SFB380/B21. References [1] E.J. Toone, E.S. Simon, G.M. Whitesides, Tetrahedron Lett. 45 (1989) 5365^5421. [2] D.G. Drueckhammer, W.J. Hennen, R.L. Pederson, C.F. Barbas, C.M. Gautheron, T. Krach, C.H. Wong, Synthesis (1991) 499^525. [3] K. Faber, in: Biotransformations in Organic Chemistry, Springer Verlag, Berlin, 1992, pp. 204^220. [4] C.H. Wong, G.M. Whitesides, in: Enzymes in Synthetic Organic Chemistry, Pergamon, Oxford, 1994, pp. 195^251. [5] C.H. Wong, R.L. Halcomb, Y. Ichikawa, T. Kajimoto, Angew. Chem. 107 (1995) 453^474. [6] H.J.M. Gijsen, L. Qiao, W. Fitz, C.H. Wong, Chem. Rev. 96 (1996) 443^473. [7] W.-D. Fessner, C. Walter, Top. Curr. Chem. 184 (1996) 97^ 194. [8] C.H. Wong, Annu. Rev. Microbiol. 51 (1997) 285^310. [9] L.O. Krampitz, Annu. Rev. Biochem. 38 (1969) 213^240. [10] D.H.G. Crout, S. Davies, R.J. Heath, C.O. Miles, R. Rathbone, B.E.P. Swoboda, M.B. Gravestone, Biocatalysis 9 (1994) 1^30. [11] German patent 548 459/1932, Verfahren zur Herstellung von 1-phenyl-2-methylaminopropan-1-ol, 1932. [12] S. Bringer-Meyer, H. Sahm, Biocatalysis 1 (1988) 321^331. [13] D.H.G. Crout, H. Dalton, D.W. Hutchinson, M. Miyagoshi, J. Chem. Soc. Perkin Trans. 1 (1991) 1329^1332. [14] M.D. Lilly, R. Chauhan, C. French, M. Gyamerah, G.R. Hobbs, A. Humphrey, M. Isupov, J.A. Littlechild, R.K. Mitra, K.G. Morris, M. Rupprecht, N.J. Turner, J.M. Ward, A.J. Willets, J.M. Woodley, Ann. NY Acad. Sci. 782 (1996) 513^525. [15] G.R. Hobbs, R.K. Mitra, R.P. Chauhan, J.M. Woodley, M.D. Lilly, J. Biotechnol. 45 (1996) 173^179. [16] U. Scho«rken, H. Sahm, G.A. Sprenger, in: H. Bisswanger, A. Schellenberger (Eds.), Biochemistry and Physiology of Thiamin Diphosphate Enzymes, A and C Intemann Verlag, Prien, 1996, pp. 543^553. [17] U. Scho«rken, Ph.D. thesis, Ju«l. Ber. No. 3418, University of Du«sseldorf, 1997.
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