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

点击翻译按钮获取中文摘要

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.