Erythromycin synthases

Fig. 5. Predicted domain organization and biosynthetic intermediates of the erythromycin synthase. Each circle represents an enzymatic domain as follows ACP, acyl carrier protein AT, acyl-transferase DH, dehydratase ER, P-ketoacyl-ACP enoyl reductase KR, [3-ketoacyl-ACP reductase KS, p-ketoacyl-ACP synthase TE, thioesterase. Zero indicates dysfunctional domain.

Long, P.F., Wilkinson, C.J., Bisang, C.P. et al. (2002) Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase. Molecular Microbiology, 43, 1215. 

Petkovic, H., Lill, R.E., Sheridan, R.M. et al. (2003) A novel erythromycin, 6-desmethyl erythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase. The Journal of Antibiotics, 56, 543. 

Ruan, X., Pereda, A., Stassi, D.L. et al. (1997) Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives. Journal of Bacteriology, 179, 6416. 

McDaniel, R., Thamchaipenet, A., Gustafsson, C. et al. (1999) Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel unnatural natural products. Proceedings of the National Academy of Sciences of the United States of America, 96, 1846. 

Gokhale, R.S., Hunziker, D., Cane, D.E. and Khosla, C. (1999) Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. Chemistry Biology, 6, 117. 

Roberts, G.A., Staunton, J. and Leadlay, P.F. (1993) Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin-producing polyketide synthase. European Journal of Biochemistry, 214, 305. 

Figure 13.1 PK synthase biosynthetic architecture. The erythromycin aglycone synthase (deoxyery-thronolide B synthase) is shown, made up of three modules with 28 domains...

It should be noted that TE-catalyzed cyclization is not Umited to the synthesis ofmacrocycUc peptides by catalyzing the formation of a C—N bond. These enzymes are also responsible for the cyclization of NRP depsipeptide and PK lactone. Indeed, a didomain excised from fengydn synthase was able to catalyze the formation of a macrolactone through the formation of a C—O bond [39]. Several cyclases from PKSs have also been characterized to be functional. For example, when a TE from picromycin synthase was fused to an erythromycin module (DEBS module 3), the resulting hybrid was able to convert a diketide and 2-methylmalonyl-CoA to a triketide ketolactone (Scheme 7.12) [40]. However, their in vitro activity is in... 

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... 

J Cortes, SH Haydock, GA Roberts, DJ Bevitt, PF Leadlay. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348 176-178, 1990. 

DJ Bevitt, J Staunton, PF Leadlay. Mutagenesis of the dehydratase active site in the erythromycin-producing polyketide synthase. Biochem Soc Trans 21 30S, 1992. 

B. Erythromycin, Rapamycin, and Other Modular Polyketide Synthases... 

R Pieper, G Luo, DE Cane, C Khosla. Cell-free biosynthesis of polyketides by recombinant erythromycin polyketide synthases. Nature 378 263-266, 1995. 

RS Gokhale, J Lau, DE Cane, C Khosla. Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase. Biochemistry 37 2524-2528, 1998. 

R Pieper, S Ebert-Khosla, DE Cane, C Khosla. Erythromycin biosynthesis kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates. Biochemistry 35 2054-2060, 1996. 

IE Holzbaur, RC Harris, M Bycroft, J Cortes, C Bisang, J Staunton, BAM Rudd, PF Leadlay. Molecular basis of Celmer s rules the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase. Chem Biol 6 189-195, 1999. 

JR Jacobsen, CR Hutchinson, DE Cane, C Khosla. Precursor directed biosynthesis of novel erythromycin analogs by an engineered polyketide synthase. Science 277 367-369, 1997. 

Figure 5 Domain organization of the erythromycin polyketide synthase. Putative domains are represented as circles and the structural residues are ignored. Each module incorporates the essential KS, AT, and ACP domains, while all but one include optional reductive activities. AT, acyltransferase ACP, acyl carrier protein KS, (3-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydratase ER, enoyl reductase TE, thioesterase.

Figure 9 Construction of bimodular polyketide synthases, (a) Chromosomal repositioning of the thioesterase domain from the C-terminus of module 6 to the end of module 2 in the erythromycin PKS leads to production of triketide lactones and the disruption of erythromycin biosynthesis, (b) DEBS 1-TE contains a fusion within the ACP domains of modules 2 and 6. In Saccharopolyspora erythraea and Streptomyces coelicolor the construct produced both propionate and acetate-derived lactones, (c) DEBS 1+TE contains a fusion between ACP2 and the thioesterase domain. In S. coelicolor, the protein biosynthesized the same lactones.

The experiments related here represent only some of the successful reengineering of the erythromycin PKS and other related synthases. In the future, this technology should provide many hybrid proteins for in vitro biosynthesis. 

KJ Weissman, M Timoney, M Bycroft, P Grice, U Hanefeld, J Staunton, PF Leadlay. The molecular basis of Celmer s rules the stereochemistry of the condensation step in chain extension on the erythromycin polyketide synthase. Biochemistry 36 13849-13855, 1997. 

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