Complete reconstitution of a highly reducing iterative polyketide synthase.
Highly reducing iterative polyketide synthases are large, multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding of the functions and structures of this family of enzymes.
Ma
Suzanne M
SM
Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA.
Li
Jesse W-H
JW
Choi
Jin W
JW
Zhou
Hui
H
Lee
K K Michael
KK
Moorthie
Vijayalakshmi A
VA
Xie
Xinkai
X
Kealey
James T
JT
Da Silva
Nancy A
NA
Vederas
John C
JC
Tang
Yi
Y
D001230:Aspergillus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000378:metabolism; D007659:Ketones / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D008148:Lovastatin / Q000096:biosynthesis; D008316:Malonyl Coenzyme A / Q000378:metabolism; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011753:Pyrones / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D005656:Fungal Proteins; D007659:Ketones; D007783:Lactones; D009097:Multienzyme Complexes; D009281:Naphthalenes; D011753:Pyrones; D011994:Recombinant Proteins; C442940:lovC protein, Aspergillus terreus; C545176:lovastatin nonaketide synthase, Aspergillus terreus; D009243:NAD; D008316:Malonyl Coenzyme A; D048630:Polyketide Synthases; D012436:S-Adenosylmethionine; C045373:dihydromonacolin L; D008148:Lovastatin