Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase

被引:73
作者
Castonguay, Roselyne
He, Weiguo
Chen, Alice Y.
Khosla, Chaitan
Cane, David E.
机构
[1] Brown Univ, Dept Chem, Providence, RI 02912 USA
[2] Stanford Univ, Dept Chem Engn, Stanford, CA 94305 USA
[3] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[4] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA
关键词
D O I
10.1021/ja0753290
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS)-acyl transferase (AT) didomains with acyl-carrier protein (ACIP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-MS technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-delta-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACPS] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACIP-bound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.
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页码:13758 / 13769
页数:12
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