Elucidating the mechanism of cis double bond formation in epothilone biosynthesis.
The epothilones, originally isolated from the myxobacterium Sorangium cellulosum, are macrocyclic compounds that are synthesized by a modular polyketide synthase, an enzyme complex composed of six large, multifunctional proteins. The penultimate intermediates in epothilone production, and the products of the PKS-catalyzed reactions, are epothilones D and C, which contain a 12,13-cis-double bond. The 12 and 13 positions of epothilones are generated during the fourth elongation step that is governed by module 4. Module 4 does not contain a dehydratase (DH) domain, which is required for dehydration to create the double bond. A DH domain, present in module 5 and presumed to act in the fifth elongation step at the 10 and 11 positions, was proposed to act as well to generate the 12,13-cis-double bond. Inactivation of the DH domain in module 5 resulted in the production of 10,11-dehydro-13-hydroxyepothilone D as the major product, confirming that DH5 is required for 12,13 dehydration. A mechanistic model based on domain skipping and modular stuttering is presented to explain the basis for the iterative DH5 activity observed.
Tang
Li
L
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA. tang@kosan.com
Ward
Shannon
S
Chung
Loleta
L
Carney
John R
JR
Li
Yong
Y
Reid
Ralph
R
Katz
Leonard
L
D004789:Enzyme Activation; D034261:Epothilones / Q000096:biosynthesis*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009231:Myxococcales / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
D034261:Epothilones; D009097:Multienzyme Complexes