The Ligase and Acetyltransferase Enzymes of Mycothiol Biosynthesis

一种新的抗氧化剂，真菌硫醇，最近被证明是由大多数放线菌产生的，放线菌是一类细菌，包含了土壤细菌的主要部分。其中包括产生疾病的分枝杆菌，如结核分枝杆菌和链菌，后者是抗生素的主要来源。真菌硫醇已被证明具有异常的抗自氧化性，并且发现耻毛分枝杆菌在真菌硫醇生物合成中受阻的化学突变体对过氧化氢的敏感性增强。这些增强的敏感性是否仅仅是由于真菌硫醇生物合成受阻，还是由于其他可能存在的突变，目前还不确定。目前的研究将验证一个假设，即菌硫醇虽然对分枝杆菌的实验室生存不是必需的，但在保护放线菌免受氧化和抗生素的攻击方面起着关键作用。真菌硫醇的生物合成涉及通过葡萄糖胺（GlcN）与肌醇（Ins）的连接组装假双糖（GlcN-Ins）。然后，半胱氨酸（Cys）通过ATP依赖性反应与GlcN-Ins结合，生成Cys-GlcN-Ins。后者被乙酰辅酶a依赖的转乙酰化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化酶步骤。将对酶进行纯化，并对其催化功能进行表征。编码催化这些反应的酶的基因将被鉴定，在这些酶中被特异性阻断的突变体将在模式生物中产生。将对这些突变体进行研究，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能力的影响。这些研究将为真菌硫醇生物合成的两个关键步骤提供基本的生化信息。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害方面的作用，并将确定真菌硫醇对抗生素的保护程度。这一结果可能对设计针对分枝杆菌的新药具有重要意义，并将为研究分枝杆菌硫醇在放线菌中的天然功能提供新的见解。

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.