Schneider Katja, Hövel Klaus, Witzel Kilian, Hamberger Björn, Schomburg Dietmar, Kombrink Erich, Stuible Hans-Peter
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8601-6. doi: 10.1073/pnas.1430550100. Epub 2003 Jun 20.
To reveal the structural principles determining substrate specificity of 4-coumarate:CoA ligase (4CL), the crystal structure of the phenylalanine activation domain of gramicidin S synthetase was used as a template for homology modeling. According to our model, 12 amino acid residues lining the Arabidopsis 4CL isoform 2 (At4CL2) substrate binding pocket (SBP) function as a signature motif generally determining 4CL substrate specificity. We used this substrate specificity code to create At4CL2 gain-of-function mutants. By increasing the space within the SBP we generated ferulic- and sinapic acid-activating At4CL2 variants. Increasing the hydrophobicity of the SBP resulted in At4CL2 variants with strongly enhanced conversion of cinnamic acid. These enzyme variants are suitable tools for investigating and influencing metabolic channeling mediated by 4CL. Knowledge of the 4CL specificity code will facilitate the prediction of substrate preference of numerous, still uncharacterized 4CL-like proteins.
为揭示决定4-香豆酸:辅酶A连接酶（4CL）底物特异性的结构原理，短杆菌肽S合成酶苯丙氨酸激活结构域的晶体结构被用作同源建模的模板。根据我们的模型，拟南芥4CL同工型2（At4CL2）底物结合口袋（SBP）内的12个氨基酸残基作为一个特征基序，通常决定4CL的底物特异性。我们使用这个底物特异性编码来创建At4CL2功能获得性突变体。通过增加SBP内的空间，我们生成了阿魏酸和芥子酸激活的At4CL2变体。增加SBP的疏水性导致At4CL2变体对肉桂酸的转化能力大幅增强。这些酶变体是研究和影响由4CL介导的代谢通道的合适工具。4CL特异性编码的知识将有助于预测众多尚未表征的4CL样蛋白的底物偏好。
