The Ligase and Acetyltransferase Enzymes of Mycothiol Biosynthesis

一种新的抗氧化剂，真菌硫醇，最近已被证明是由大多数放线菌，一类细菌，其中包括一个主要部分的土壤细菌。 这些包括致病的分枝杆菌，如结核分枝杆菌，以及抗生素的主要来源链霉菌。 霉硫醇已被证明对自氧化和M的化学突变体具有异常的抗性。已发现在真菌硫醇生物合成中被阻断的丝霉烯对过氧化氢表现出增强的敏感性。 这些增强的敏感性是否仅仅是由于真菌硫醇生物合成的阻断或可能存在的其他突变引起的尚不确定。 目前的研究将测试的假设，虽然不是必不可少的实验室生存的分枝杆菌，在保护放线菌对氧化和抗生素的挑战，发挥了关键作用。真菌硫醇生物合成涉及通过葡糖胺（GlcN）与肌醇（Ins）的连接组装假二糖（GlcN-Ins）。 然后，半胱氨酸（Cys）在ATP依赖性反应中与GlcN-Ins连接以产生Cys-GlcN-Ins。后者被乙酰辅酶A依赖性转乙酰酶乙酰化以产生真菌硫醇（AcCys-GlcN-Ins）。 本研究将探讨真菌硫醇生物合成的连接酶和转乙酰酶步骤。 酶将被纯化并表征其催化功能。 将鉴定编码催化这些反应的酶的基因，并在模式生物M中产生特异性阻断这些酶的突变体。 恶臭 将研究这些突变体以确定阻断真菌硫醇生物合成对生物体抵抗氧化和抗生素应激的能力的影响。这些研究将提供有关真菌硫醇生物合成中两个关键步骤的基本生物化学信息。 他们还将测试真菌硫醇在保护分枝杆菌免受过氧化氢和其他氧化剂损伤中的作用，并将确定真菌硫醇可以保护抗生素的程度。 这些结果可能在设计针对分枝杆菌的新药方面具有重要意义，并将提供有关放线菌中真菌硫醇天然功能的见解。

A novel antioxidant, mycothiol, has recently been shown to be produced by most actinomycetes, a class of bacteria encompasing a major fraction of soil bacteria. These include disease-producing mycobacteria, such as Mycobacterium tuberculosis, and the streptomycetes, a major source of antibiotics. Mycothiol has been shown to be unusually resistant to autooxidation and chemical mutants of M. smegmatis blocked in mycothiol biosynthesis have been found to exhibit enhanced sensitivity to hydrogen peroxide. Whether these enhanced sensitivities result solely from blockage of mycothiol biosynthesis or from other mutations which might also be present is uncertain. The present studies will test the hypothesis that mycothiol, although not essential for laboratory survival of mycobacteria, plays a key role in protecting actinomycetes against oxidative and antibiotic challenge. Mycothiol biosynthesis involves the assembly of a pseudodisaccharide (GlcN-Ins) by linking of glucosamine (GlcN) with myo- inositol (Ins). Cysteine (Cys) is then ligated to GlcN-Ins in an ATP- dependent reaction to produce Cys-GlcN-Ins. The latter is acetylated by a acetyl CoA-dependent transacetylase to produce mycothiol (AcCys-GlcN-Ins). This research will investigate the ligase and transacetylase steps of mycothiol biosynthesis. The enzymes will be purified and characterized with respect to their catalytic functions. The genes coding the enzymes catalyzing these reactions will be identified and mutants blocked specifically in these enzymes will be produced in the model organism M. smegmatis . These mutants will be studied to ascertain the effects of blocking mycothiol biosynthesis upon the ability of the organism to resist oxidative and antibiotic stress. These studies will provide fundamental biochemical information about two key steps in the biosynthesis of mycothiol. They will also test the role of mycothiol in the protection of mycobacteria against damage by hydrogen peroxide and other oxidants, and will establish the extent to which mycothiol can protect against antibiotics. The results could prove important in the design of new drugs directed against mycobacteria and will provide insight concerning the natural function of mycothiol in actinomycetes.