6-deoxyerythronolide B synthase DEBS

Figure 12.5 A. Comparison of the CHS monomer (left) and P-ketoacyl synthase monomer (right). The structurally conserved secondary structure of each monomer s upper domain is colored in blue (a-helix) and gold (P-strand). Portions of each protein monomer forming the dimer interface are colored purple. The side-chains of the catalytic residues of CHS (Cysl64, His303, Asn336) and P-ketoacyl synthase (Cysl63, His303, His340) are shown. B. Sequence conservation of the catalytic residues of CHS, 2-PS, p-ketoacyl synthase (FAS II), and the ketosynthase modules of 6-deoxyerythronolide B synthase (DEBS), actinorhodin synthase (ActI) and tetracenomycin synthase (TcmK). The catalytic residues are in red.

Deoxyerythronolide B synthase (DEBS) is a modular Type I PKS involved in erythromycin biosynthesis (see page 96) and its structure and function are illustrated in Figure 3.85. The enzyme contains three subunits (DEBS-1, 2, and 3), each encoded by a gene (eryA-l, II, and III). It has a linear organization of six modules, each of which... 

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... 

Figure 10.1 Domain and modular structure of 6-deoxyerythronolide B synthase (DEBS), the model modular PKS. ACP = acyl carrier protein ...

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. 

These studies led to the classification of PKSs into two major types, type I and type II. The type I enzymes are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of 6-deoxyer-ythronolide B (6-dEB), the macrolactone... 

Fig. 9.2-3 Schematic diagram of 6-deoxyerythronolide B synthase (DEBS) The synthase consists of three separate polypeptides composed of two modules each, which are responsible for a single ketide-unit elongation of the 6-deoxyerythronolide B backbone. The...

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Biosynthesis of erythromycin represents a typical polyketide pathway and structural organization of PKSs. Erythromycin PKS (6-deoxyerythronolide B synthase, DEBS) uses propionyl-CoA as a priming unit and six methylmalonyl-CoA molecules as extender units. Each module in a modular PKS contains domains for one round of chain elongation (KS, AT, ACP) and p-keto modification (ketoreductase, KR dehy-drase, DH enoylreductase, ER). DEBS consists of six modules (mod 1 -6) encoded on three separate polypeptide subunits. 

The initial assembly of seven acyl CoA precursors to build a polyketide carbon chain is carried out by a multienzyme complex called a polyketide synthase, or PKS. The 6-deoxyerythronolide B synthase (DEBS) is a massive structure of greater than 2 million molecular weight and containing more than 20,000 amino acids. Furthermore, it is a homodimer, meaning that it consists... 

FIGURE 25.17 A schematic view ofthe 6-deoxyerythronolide B synthase (DEBS), showing the locations ofthe enzyme domains within the loading module and the six extension modules. The figure is explained in detail in the text. 

An early example in which such a bioengineering approach has been successfully attempted is the bioengineering of the 6-deoxyerythronolide B synthase (DEBS) [45]. The DEBS multienzyme complex consists of 3 large subunits (>300 kDa), each containing two modules (in total 28 catalytic domains). DNA manipulations were performed in Escherichia coli XLl and Streptomyces coelicolor or lividans were used as the host for the production of polyketides. 50 compounds were produced in yields of 0.005-0.7% (relative to 6dEB under the same conditions) (Eig. 15.2). This was an impressive example for the potential power of this strategy, but it fades when one considers that theoretically the DEBS gene cluster could be permutated with the result of 10 different compounds. 

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