Essential role of the donor acyl carrier protein in stereoselective chain translocation to a fully reducing module of the nanchangmycin polyketide synthase.
Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min⁻¹ and a k(cat)/K(m) of 67 min⁻¹ mM⁻¹, corresponding to a ∼2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2.
Department of Chemistry, Brown University, Providence, Rhode Island 02912-9108, United States.
Xun
X
Guo
Tiangang
T
Liu
Zixin
Z
Deng
David E
DE
Cane
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000215:Acylation; D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D004987:Ethers / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004987:Ethers; D061065:Polyketides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D013141:Spiro Compounds; C452419:nanchangmycin; C000904:dianemycin; D048630:Polyketide Synthases