Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany.
INCITAP-CONICET, Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de La Pampa, Avenida Uruguay 151, 6300, Santa Rosa, La Pampa, Argentina.
Chembiochem. 2019 Jul 1;20(13):1672-1677. doi: 10.1002/cbic.201900050. Epub 2019 May 24.
Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg-Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.
分支点中间体分支点中间体在微生物和植物中许多芳香族代谢物的生物合成中构成分支点中间体。为了获得非天然化合物，我们修饰了大肠杆菌中甲羟戊酸途径。我们提出了一个异分支点与 MenD（（1R，2S，5S，6S）-2-琥珀酰基-5-烯醇丙酮酰基-6-羟基环己-3-烯-1-羧酸酯（SEPHCHC）合酶）的活性位点结合的模型，该模型解释了与α-酮戊二酸的天然反应的结果。我们已经合理设计了 MenD 的变体，用于几种异分支点类似物的转化。双变体 Asn117Arg-Leu478Thr 优先转化（5S，6S）-5,6-二羟基环己-1,3-二烯-1-羧酸（2,3-trans-CHD），异分支点的水解产物，比野生型高 >70 倍。单变体 Arg107Ile 使用（5S，6S）-6-氨基-5-羟基环己-1,3-二烯-1-羧酸（2,3-trans-CHA）作为底物，与野生型 MenD 相比转化率 >6 倍。这些新化合物已经在体内获得（高达 5.3 g/L）。出乎意料的是，由于鉴定出的残基（如 Arg107）高度保守（>94%），因此在其他细菌的野生型 SEPHCHC 合酶中也可以发现一些设计的变化（Arg107Lys，0.3%）。这就提出了一个问题，即尚待探索的莽草酸途径分支是否可能自然发生。
