Deoxyerythronolide synthase

The striking similarity of the overall topology of the KS-AT didomain of the PKS 6-deoxyerythronolide synthase, the most studied PKS, with its FAS counterpart, also contributes important information on the structure. Exciting new structural data on the FAS systems in mycobacteria, mammals, yeast, and fungi have recently been reported [10-13], and these efforts have been thoroughly reviewed [14],... 

Wang, Y. and Pfeifer, B.A. (2007) 6-Deoxyerythronolide B production through chromosomal localization ofthe deoxyerythronolide B synthase genes in E. coli. Metabolic Engineering, 10, 33-38. 

Ward, S.L., Desai, R.P., Hu, Z. et al. (2007) Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase. Journal of Industrial Microbiology and Biotechnology, 34, 9-15. 

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.

Figure 21-11 Catalytic domains within three polypeptide chains of the modular polyketide synthase that forms 6-deoxyerythronolide B, the aglycone of the widely used antibiotic erythromycin. The domains are labeled as for fatty acid synthases AT, acyltransferase ACP, acyl carrier protein KS, 3-ketoacyl-ACP synthase KR, ketoreductase DH, dehydrase ER, enoylreductase TE, thioesterase. After Pieper et al.338 Courtesy of Chaitan Khosla.

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... 

DJ Bevitt, J Cortes, SF Haydock, PF Leadlay. 6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme. Eur J Biochem 204 39-49, 1992. 

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. 

Tang Y, Kim CY, Mathews II, Cane DE, Khosla C (2006) The 2.7-Angstrom Crystal Structure of a 194-kDa Homodimeric Fragment of the 6-Deoxyerythronolide B Synthase. Proc Natl Acad Scl USA 103 11124... 

The biosynthesis of erythromycin can be divided into two phases (Scheme 1). In the first constructive phase of the pathway a set of key enzymes, collectively known as the polyketide synthase (PKS), assembles the typical polyketide chain by sequential condensation of one unit of propionyl-CoA and six units of methylmalonyl-CoA 6. The initially formed chain is cyclised to give the first macrocychc lactone (macrolide) intermediate 6-deoxyerythronolide B 7 [6,7]. In the second phase 6-deoxyerythronohde B is elaborated by a series of tailoring enzymes which carry out regiospecific hydroxylations, glycosylations and a methylation (of an added sugar residue) to give finally erythromycin A. The core polyketide structure is generated by the PKS in phase one, but the later steps of phase two are essential to produce active antibiotics. 

Figure 2.7 Domain organisation of erythromycin polyketide synthase. Gene sequence putative domains are represented as circles and the structural residues are ignored. Each module incorporates the essential KS, AT andACP domains, while all but one include optional reductive activities (KR, DH, ER). The one-to-one correspondence between domains and biosynthetic transformations explains how programming is achieved in this modular PKS. DEBS—deoxyerythronolide B synthase (reproduced with permission of Prof. James Staunton)...

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... 

Jacobsen, J.R., Cane, D.E. Khosla, C. Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to poly-ketide biosynthesis. Biochemistry 37, 4928-4934 (1998). 

Roberts, G.A., Staunton, J. Leadlay, P.F. 6-Deoxyerythronolide B synthase 3 from Saccharopolyspora erythraea over-expression in... 

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