title: Spontaneous priming of a downstream module in 6-deoxyerythronolide B synthase leads to polyketide biosynthesis.
abstract: Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Jacobsen
authors.forename: D E
authors.initials: DE
authors.lastname: Cane
authors.forename: C
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000595:Amino Acid Sequence; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C034277:6-deoxyerythronolide B; D004917:Erythromycin