MenD (SEPHCHC synthase), a thiamine-dependent enzyme with similarity to the Stetterases

作为细菌甲基萘醌生物合成途径中唯一的硫胺素二磷酸依赖酶，MenD （SEPHCHC合成酶）在既不显示醛也不显示酮结构的环结构受体化合物上进行1,4加成反应。此外，Michael m<e:1> ller的工作小组在芳醛上发现了一个有趣的1,2添加物，从而允许合成新的2-羟基酮。除了a-酮戊二酸（a-氧戊二酸）外，MenD还可以利用丙酮酸或草酰乙酸作为供体化合物。我们在项目框架内的工作目的是更详细地分析MenD的底物光谱（包括供体侧和受体侧），并使酶适用于制备性合成。基于酶的三维结构-最近可用-我们希望与建模小组合作，研究酶活性位点所有氨基酸残基的功能。特别强调的是参与底物结合和/或催化1,2和1,4添加的残基。使用位点定向饱和诱变，我们将瞄准具有改变底物特异性的酶变体。此外，抑制MenD活性的底物类似物可能为新型抗生素提供有趣的先导结构，因为甲基萘醌生物合成途径对几种致病菌（如结核分枝杆菌）至关重要。

As the only thiamine diphosphate-dependent enzyme, MenD (SEPHCHC synthase) from the bacterial menaquinone biosynthetic pathway, performs an 1,4 addition reaction on a ring structured acceptor compound which neither displays an aldehyde nor a keto structure. Moreover, the working group of Michael Müller has discovered an interesting 1,2 addition on arylaldehydes, thus allowing the synthetic access to novel 2-hydroxy ketones. Besides a-ketoglutarate (a-oxoglutarate), MenD is able to utilize pyruvate or oxaloacetate as donor compounds. The aim of our work in the frame of the project is to analyze the substrate spectrum (both donor and acceptor side) of MenD in more detail and to make the enzyme applicable in preparative syntheses. Based on the 3D structure of the enzyme - which became available recently- we want to cooperate with a modeling group and study the function of all amino acid residues of the enzyme s active site. Special emphasis will be on residues which are involved in substrate binding and/or the catalysis of 1,2 and 1,4 additions. Using site-directed saturation mutagenesis, we will aim for enzyme variants with altered substrate specificities. As well, substrate analogues which inhibit MenD activity may provide interesting lead structures for novel antibiotics as the menaquinone biosynthetic pathway is essential for several pathogenic bacteria, e.g. Mycobacterium tuberculosis.