Lu Hongxiang, Tsai Shiou-Chuan, Khosla Chaitan, Cane David E
Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA.
Biochemistry. 2002 Oct 22;41(42):12590-7. doi: 10.1021/bi026006d.
The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.
甲基霉素/苦霉素合酶（PICS）的硫酯酶（TE）结构域在大肠杆菌中实现了功能表达，并确定了重组TE的最佳N端边界。测试了一系列二酮基-N-乙酰半胱胺（SNAC）硫酯作为底物。与还原二酮类化合物1-4的立体异构体相比，PICS TE对2-甲基-3-氧代戊酰-SNAC底物5表现出强烈的偏好，对（2R,3S）-2-甲基-3-羟基非对映异构体2的偏好约为（2S,3R）-二酮类化合物1的1.6:1。密切相关的DEBS TE，即6-脱氧红霉内酯B合酶的硫酯酶，对2的偏好更为明显，为4.4:1，对3-氧代酰基-SNAC底物5的偏好仅略高。通过定点诱变研究了PICS TE中几个活性位点残基的作用。丝氨酸148是由S148、H268和D176组成的明显催化三联体的一部分，发现它对硫酯酶活性至关重要，而用天冬酰胺（D176N）取代D176得到的突变硫酯酶对二酮基-SNAC底物仍保留大量（尽管有所降低）的水解活性。E187和R191各自被认为在底物结合中起作用，它们的突变对二酮基底物1、2和5的相对特异性只有轻微影响。最后，当PICS TE与DEBS模块3的C端融合时，与先前开发的DEBS模块3-（DEBS）TE构建体相比，所得嵌合蛋白将二酮类化合物1与甲基丙二酰辅酶A转化为三酮内酯6，催化效率得到提高。
