CHARACTERIZATION OF THE ENZYMATIC DOMAINS IN THE MODULAR POLYKETIDE SYNTHASE INVOLVED IN RIFAMYCIN-B BIOSYNTHESIS BY AMYCOLATOPSIS-MEDITERRANEI

Five clustered polyketide synthase (PKS) genes, rifA-rifE, involved in rifamycin (Rf) biosynthesis in Amycolatopsis mediterranei S699 have b een cloned and sequenced (August, P.R. et al., 1998. Chem Biol. 5, 69- 79). The five multifunctional polypeptides constitute a type I modular PKS that contains ten modules, each responsible for a specific round of polyketide chain elongation. Sequence comparisons of the Rf PKS pro teins with other prokaryotic modular PKSs elucidated the regions that have an important role in enzyme activity and specificity. The beta-ke toacyl:acyl carrier protein synthase (KS) domains show the highest deg ree of similarity between themselves (86-90%) and to other PKSs (78-85 %) among all the constituent domains. Both malonyl-coenzyme A (MCoA) a nd methylmalonyl-coenzyme A (mMCoA) are substrates for chain elongatio n steps carried out by the Rf PKS. Since acyltransferase (AT) domains of modular PKSs can distinguish between these two substrates, comparis on of the sequence of all ten AT domains of the Rf PKS with those foun d in the erythromycin (Er) (Donadio, S. and Katz, L., 1992. Gene 111, 51-60) and rapamycin (Rp) (Haydock, S. et al., 1995. FEBS Lett. 374, 2 46-248) PKSs revealed that the AT domains in module 2 of RifA and modu le 9 of RifE are specific for MCoA, whereas the other eight modules sp ecify mMCoA. Dehydration of the beta-hydroxyacylthioester intermediate s should occur during the reactions catalysed by module 4 of RifB and modules 9 and 10 of RifE, yet only the active site region of module 4 conforms closely to the dehydratase (DH) motifs in the Er and Rp PKSs. The DH domains of modules 9 and 10 diverge significantly from the con sensus sequence defined by the Er and Rp PKSs, except for the active s ite His residues. Deletions in the DH active sites of module I in RifA and module 5 in RifB and in the N- and C-terminal regions of module 8 of RifD should inactivate these domains, and module 2 of RifA lacks a DH domain, all of which are consistent with the proposed biosynthesis of Rf. In contrast, module 6 of RifB and module 7 of RifC appear to c ontain intact DH domains even though DH activity is not apparently req uired in these modules. Module 2 of RifA lacks a beta-ketoacyl:acyl ca rrier protein reductase (KR) domain and the one in module 3 has an app arently inactive NADPH binding motif, similar to one found in the Er P KS, while the other eight KR domains of the Rf PKS should be functiona l. These observations are consistent with biosynthetic predictions. Al l the acyl carrier protein (ACP) domains, while clearly functional, ne vertheless have active site signature sequences distinctive from those of the Er and Rp PKSs. Module 2 of RifA has only the core domains (KS , AT and ACP). The starter unit ligase (SUL) and ACP domains present i n the N-terminus of RifA direct the selection and loading of the start er unit, 3-amino-5-hydroxybenzoic acid (AHBA), onto the PKS. AHBA is m ade by the products of several other genes in the Rf cluster through a variant of the shikimate pathway (August, P.R. et al., inter alia). R ifF, produced by the gene immediately downstream of rifE, is thought t o catalyse the intramolecular cyclization of the PKS product, thereby forming the ansamacrolide precursor of Rf B. (C) 1998 Elsevier Science B.V. All rights reserved.