6-deoxyerythronolide B synthase 1 is specifically acylated by a diketide intermediate at the beta-ketoacyl-acyl carrier protein synthase domain of module 2.
We have used 6-deoxyerythronolide B synthase (DEBS) as a model system to investigate molecular recognition by a modular polyketide synthase (PKS). DEBS consists of three proteins (DEBS1, -2, and -3) that biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Active sites within these multifunctional proteins are organized into biosynthetic "modules", each of which catalyzes a discrete round of polyketide chain elongation and adjusts the appropriate level of beta-ketoacylthioester reduction. Using DEBS1, we demonstrate that there is a substantial degree of molecular recognition in the processing of the natural diketide chain elongation intermediate. Exogenously added (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester is exclusively recognized by its cognate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS2). Labeled diketide specifically acylated DEBS1 in crude protein extracts and limited proteolysis localized the binding to module 2. The precise site of acylation in DEBS1 was established by the finding that a Cys2200 Ala mutant of DEBS1, lacking the KS2 active-site cysteine, did not undergo acylation by the diketide. Pretreatment of the wild-type protein with the beta-ketoacyl-ACP synthase inhibitor cerulenin also blocked acylation. These results indicate that in addition to the purely organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition. This conclusion has important implications for the use of PKSs to rationally design novel polyketides.
Tsukamoto
N
N
Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA.
Chuck
J A
JA
Luo
G
G
Kao
C M
CM
Khosla
C
C
Cane
D E
DE
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000378:metabolism*; D000215:Acylation; D000595:Amino Acid Sequence; D002569:Cerulenin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D013302:Streptomyces
D009097:Multienzyme Complexes; D002569:Cerulenin; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase