The Ligase and Acetyltransferase Enzymes of Mycothiol Biosynthesis

一种新的抗氧化剂，霉硫醇，最近被证明是由大多数放线菌产生的，放线菌是土壤细菌中的一大类。其中包括引起疾病的分枝杆菌，如结核分枝杆菌，以及链霉菌，这是抗生素的主要来源。霉菌硫醇被证明对自氧化具有异常的抗性，而阻止霉菌硫醇生物合成的耻垢分枝杆菌的化学突变体被发现对过氧化氢表现出更高的敏感性。这些增强的敏感性是否仅仅是由于霉菌硫醇生物合成的阻断或也可能存在的其他突变所致尚不确定。目前的研究将检验这一假设，即真菌硫醇虽然不是分枝杆菌在实验室生存所必需的，但在保护放线菌免受氧化和抗生素挑战方面发挥着关键作用。霉硫醇的生物合成涉及通过氨基葡萄糖(GlcN)和肌醇(INS)的连接来组装假二糖(GlcN-ins)。半胱氨酸(Cys)与谷氨酸氨基转移酶(GlcN-ins)在ATP依赖的反应中连接，生成Cys-GlcN-ins。后者被乙酰辅酶A依赖的转乙酰基酶乙酰化，生成霉菌硫醇(AcCys-GlcN-ins)。本研究将对霉菌硫醇生物合成的连接酶和转乙酰基酶步骤进行研究。这些酶将被提纯，并根据其催化功能进行表征。将识别催化这些反应的酶的编码基因，并在模式生物耻垢分枝杆菌中产生在这些酶中被特异性阻止的突变。将对这些突变体进行研究，以确定阻止霉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。这些研究将提供有关霉菌硫醇生物合成的两个关键步骤的基本生化信息。他们还将测试霉菌硫醇在保护分枝杆菌免受过氧化氢和其他氧化剂破坏方面的作用，并将确定霉菌硫醇对抗生素的保护程度。这一结果可能在针对分枝杆菌的新药设计中被证明是重要的，并将为了解放线菌中分枝杆菌硫醇的自然功能提供洞察。

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.