Reductases ketoreductase

D. Dehydratase, Enoyl Reductase, Ketoreductase, and Thioesterase Domains... 

In a study aim to develop biocatalytic process for the synthesis of Kaneka alcohol, apotential intermediate for the synthesis of HMG-CoA reductase inhibitors, cell suspensions of Acine-tobacter sp. SC 13 874 was found to reduce diketo ethyl ester to give the desired syn-(AR,5S)-dihydroxy ester with an ee of 99% and a de of 63% (Figure 7.4). When the tert-butyl ester was used as the starting material, a mixture of mono- and di-hydroxy esters was obtained with the dihydroxy ester showing an ee of 87% and de of 51% for the desired, sy -(3/t,5,Sr)-dihydroxy ester [16]. Three different ketoreductases were purified from this strain. Reductase I only catalyzes the reduction of diketo ester to its monohydroxy products, whereas reductase II catalyzes the formation of dihydroxy products from monohydroxy substrates. A third reductase (III) catalyzes the reduction of diketo ester to, vv -(3/t,55)-dihydroxy ester. 

All polyketides use the same general mechanism for chain elongation. Acetyl coenzyme A provides acetate (C2) units, which are condensed by a ketosynthase (KS). This in turn catalyzes condensation of the growing chain onto an acyl carrier protein (ACP), as generalized in Fig. 1.4. Enzymes such as ketoreductase (KR), enoyl reductase (ER), and dehydratase (DH) establish the oxidation state of caibon during translation, imparting structural diversity. Successive translation of each module leads to a chain of the required length that is eventually passed to thioeste-rase (TE), which releases the chain as a free acid or lactone. 

Scheme 23 Example of an acyl carrier protein (ACP in red) in a type I FAS. The palmitic acid is depicted as a representative fatty acid. During its biosynthesis, the ACP (red) interacts iteratively with each domain (DH, dehydrogenase ER, enoyl reductase KR, ketoreductase KS, ketosynthase TE, thioesterase) until the palmitic acid has reached its proper length.

FIGURE 19.4 Modular organization of the six modules (I—VI) of 6-deoxyerythronolide B synthase (DEBS) enzyme as derived from Saccharopolyspora erythraea. Enzyme activities are acyltransferases (AT), acyl carrier proteins (ACP), fi-ketoacyl-ACP synthases (KS), P-ketoreductases (KR), dehytratases (DH), enoyl reductases (ER), and thioesterases (TE). The TE-catalyzed release of the polyketide chain results in the formation of 6-dEB (70), 375 379 383... 

Three different ketoreductases were purified to homogeneity, and their biochemical properties were compared. Reductase I only catalyzes the reduction of ethyl diketoester 39 to its monohydroxy products 42 and 43 whereas reductase II catalyzes the formation of dihydroxy products from monohydroxy substrates. A third reductase (III) was identified which catalyzes the reduction of diketoester 39 to desired. vv -(3.R,5S)-dihydroxy ester 40a (Guo el al., 2006b). 

There is, however, a further group of NADPH-dependent carbonyl reductases that transfer the 4-pro-S hydrogen [143]. These include human brain aldehyde reductase I [144], liver xenobiotic ketone reductase [145] and prostaglandin 9-ketoreductase [146], all monomeric proteins with molecular weights in the range of 30000-400000. 

AT = acyl transferase DH = dehydratase ER = enoyl reductase KR = ketoreductase KS = ketosynthase mAT = methylmalonyl-specific acyl transferase. 

Figure 10.2 The PKS/NRPS biosynthetic paradigm, showing the most common domains and their relative positions within a modular PKS/NRPS enzyme. A = adenylation AT = acyl transferase C = condensation DH = dehydratase Ep = epimerase ER = enoyl reductase KR = ketoreductase KS = ketosynthase MT = methyltransferase PCP = peptidyl carrier protein TE = thioesterase.

The )9-ketoacyl-synthases/acyltransferases (KS/ AT) in each module effect the chain elongation by methyl-malonyl-coenzyme A units catalyzing a Claisen e.ster condensation followed by decarboxylation (Scheme 2). Subsequent domains are module-specific ketoreductases (KR), dehydratases (DH) or enoyl-reductases (ER), which regulate the functionalization of the newly prepared fi-oxoesters. The stepwise growing chain is picked up by an acyl-carrier protein (ACP). 

Modular PKS enzymes are responsible for the synthesis of a wide diversity of structures and seem to have more relaxed specificities in several of the enzymatic steps. Their enormous appeal for combinatorial purposes, though, derives from the presence of multiple modules that can be manipulated independently, allowing the production of rings of different sizes and with potential stereochemical variation at each PK carbon. The higher complexity of these pathways has somewhat hindered their exploitation, but recently, several have been fully characterized. Among them, by far the most studied modular multienzyme complex is 6-deoxyerythronolide B synthase (DEBS 240,266,267), which produces the 14-member macrolide 6-deoxyerythronolide B (10.70, Fig. 10.45). DEBS contains three large subunits each of which contains two PKS enzyme modules. Each module contains the minimal PKS enzyme vide supra) and either none (M3), one (ketoreductase KR Ml, M2, MS, and M6), or three (dehydratase DH-enoyl reductase ER-ketoreductase KR, M4) catalytic activities that produce a keto (M3), an hydroxy (Ml, M2, MS and M6), or an unsubstituted methylene (M4) on the last monomeric unit of the growing chain (Fig. 10.45). A final thioesterase (TE) activity catalyzes lactone formation with concomitant release of 10.70 from the multienzyme complex. Introduction of TE activity after an upstream module allows various reduced-size macrolides (10.71-10.73, Eig. 10.45) to be obtained. 

The seven activities of animal FASs are encoded as separate domains of a single 250 kDa polypeptide (Fig. 2) [30, 31]. These include a -ketoacyl synthase, malonyl/acetyl transferase, -ketoreductase, dehydratase, enoyl reductase, and an ACP domain with a phosphopantetheinylated serine. In addition to these activities, the animal FAS also includes a thioesterase domain which cleaves the product fatty acid from the enzyme. Proteolytic mapping of the polypeptide and genetic analysis have defined the location of the various domains in the primary sequence [30,31]. 

Figure 6 HMGS containing biosynthetic pathways. Portions of the PKS and PKS/NRPS pathways where the HMGS and related enzymes are located. Abbreviations A - Adenylation, AGP - acyl carrier protein, AT - acyltransferase, Cy - cyciization, DH - dehydratase, ER - enoyl reductase, GNAT -CCN5-related N-acetyltransferase, KS - ketosynthase, KR - ketoreductase, MT - methyltransferase. Ox - Oxidase, Oxy - Oxygenase, PGP - peptide carrier protein, PhyH - phytanoyl-CoA dioxygenase, PS - pyrone synthase, TE - thioesterase, - unknown function, - inactive domain.

Figure 3. Relationship between polyketide and fatty acid biosynthesis. The simplest ( minimaV) PKSs possess ketosynthase activity and produce linear polyketide products. In contrast, FASs also catalyze successive ketoreduction-dehydration-enoyl reduction reactions following each condensation. Diverse PKSs may perform none, part, or all of this reductive sequence. KS, ketosynthase KR, ketoreductase DH, dehydratase ER, enoyl reductase.

Figure 4. Organization of representative type 1, II, and III polyketide synthases. Upper modular arrangement of DEBS 1,2,3 subunits Center orientation and arrangement of open reading frames in actinorhodin gene cluster Lower chalcone synthase subunit. AT, acyltransferase ACP, acyl carrier protein KS, ketosynthase KR, ketoreductase DH, dehydratase ER, enoyl reductase TE, thioesterase TA, tailoring enzyme R/T, regulatory/transport related AR aromatase CY, cyclase.

Figure I. Schematic representation of domain architecture in Jungal Type I PKSs based on reported gene sequences. KS. ketoacyl, AT, acyltransferase DH, def dratase KR. ketoreductase ER, enoyl reductase MT, methyltransferase CLC, Claisen-iike cyclase MSAS, methylsalicylic acid synthase THN, tetrahydronaphthalene AF, ST, ( atoxin, sterigmatocystin...

Figure 2. (A) Structures of aromatic polyketides produced by A. arborescens. (B) Khellin andvisnagin in Ammi visnaga. (C) A hypothetical scheme for the involvement of OKS and as yet unidentified ketoreductase in the biosynthesis of anthrones and anthraquinones. In the absence of interactions with the reductase, OKS Just affords SEK4/SEK4b as shunt products, (Adapted from reference 7b, Copyright 2005 American Chemical Society,)...

Many of the above studies have given invaluable information on the stereochemical outcome of the ketoreductase and dehydratase catalysed reactions occurring during polyketide assembly in fungi. A number of studies of incorporation of [2H3]acetate have provided indirect information on the stereochemistry of the final enoyl reductase reaction. Thus 2H label is found at the pro-... 

Figure 1 Hypothetical pentaketide biosynthetic system, which illustrates the enzymatic logic of type I modular polyketide synthases (PKSs) and the catalytic role of acyl transferase (AT) domains. Each AT domain selects substrates from the cellular pool and tethers them as thioesters to acyl carrier protein (ACP) domains. In a typical PKS module, the AT and ACP domains are present in all modules. The ketosynthase (KS) domain is present in all chain extension modules. The dehydratase (DH), enoyl reductase (ER), and ketoreductase (KR) domains are optional domains. The final thioesterase (TE) domain catalyzes the release of the product from the PKS.

E. coli fell into the PKS associated gene. These included an AGP, four /3-ketoacyl AGP synthases, an aminotransferase, an AGP reductase, and a ketoreductase (Figure 9). Two transcription factors (a LysR-type response regulator and a IclR-type response regulator) located adjacent to the predicted biosynthetic genes... 

Besides these examples, many other important enzymes for biocatalytic reductions, such as the NADPH-dependent carbonyl reductase from Candida magnoliae U2 the ketoreductase from Zygosaccharomyces rowxii11431, and the aldehyde reductase from Sporobolomyces salmonicolor AKU442911441, etc. have also been expressed in E. coli etc. and shown to be active. 

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