Molecular basis of Celmer's rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases.
A system is reported for the recombinant expression of individual ketoreductase (KR) domains from modular polyketide synthases (PKSs) and scrutiny of their intrinsic specificity and stereospecificity toward surrogate diketide substrates. The eryKR(1) and the tylKR(1) domains, derived from the first extension module of the erythromycin PKS and the tylosin PKS, respectively, both catalyzed reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, with complete stereoselectivity and stereospecificity, even though the substrate is not tethered to an acyl carrier protein or an intact PKS multienzyme. In contrast, and to varying degrees, the isolated enzymes eryKR(2), eryKR(5), and eryKR(6) exercised poorer control over substrate selection and the stereochemical course of ketoreduction. These data, together with modeling of diketide binding to KR(1) and KR(2), demonstrate the fine energetic balance between alternative modes of presentation of ketoacylthioester substrates to KR active sites.
Siskos
Alexandros P
AP
Department of Biochemistry, University of Cambridge, UK.
Baerga-Ortiz
Abel
A
Bali
Shilpa
S
Stein
Viktor
V
Mamdani
Hassan
H
Spiteller
Dieter
D
Popovic
Bojana
B
Spencer
Jonathan B
JB
Staunton
James
J
Weissman
Kira J
KJ
Leadlay
Peter F
PF
D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013379:Substrate Specificity
D048630:Polyketide Synthases; D010088:Oxidoreductases