ORGANIZATION OF THE BIOSYNTHETIC GENE-CLUSTER FOR RAPAMYCIN IN STREPTOMYCES-HYGROSCOPICUS - ANALYSIS OF THE ENZYMATIC DOMAINS IN THE MODULAR POLYKETIDE SYNTHASE

The three giant multifunctional polypeptides of the rapamycin (Rp)-pro ducing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowe d further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), beta-ketoacyl-ACP syntha se (KS) and acyltransferase (AT) domains (the core domains) have an ex tremely high degree of mutual sequence homology. The KS domains in par ticular are almost perfect repeats over their entire length, Module 14 shows the least homology and is unique in possessing only core domain s, The enoyl reductase (ER), beta-ketoacyl-ACP reductase (KR) and dehy dratase (DH) domains are present even in certain modules where they ar e not apparently required, Four DH domains can be recognised as inacti ve by characteristic deletions in active site sequences, but for two o thers, and for KR and ER in module 3, the sequence is not distinguisha ble from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity t hat may modify the starter unit after attachment, The sequence compari son has revealed the surprisingly high sequence similarity between int er-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisat ion into active species.