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首页> 外文期刊>Biochemistry >Characterization of the N-Acetyl-α-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis
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Characterization of the N-Acetyl-α-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis

机译：N-乙酰基-α-d-氨基葡萄糖氨基乙酸苹果酸合酶和去乙酰化酶功能在炭疽芽孢杆菌中的杆菌硫醇生物合成

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摘要

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weightrnthiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lackingrnglutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosyntheticrnpathway in B. anthracis, which combine to produce R-D-glucosaminyl L-malate (GlcN-malate) from UDP-rnGlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have thernkinetic parameters for the BaBshA glycosyltransferase (fGlcNAc-malate) and the BaBshB deacetylasern(fGlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and therncrystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 A ˚rnresolution,rnidentifies several active-site interactions important for the specific recognition of L-malate, but not otherrnR-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-rnmyo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is nomajor conformationalrnchange observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracisrndeficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locusrn(BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, orrngermination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M.rn(2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for therndevelopment of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens likernB. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not wellrnunderstood, and we present a composite picture based on this and other recent workthese ligands can effectively induce G-quadruplexnstructures in the G-rich 22-mer c-MYC DNA sequence and further stabilize the structure. Equilibriumnbinding constantsmeasured by isothermal titration calorimetermethods indicate a high affinity of the ligandsnfor the quadruplex structures (K ∼ 107nM-1n) and no affinity for the duplex DNA, demonstrating that thesenligands are selective for G-quadruplex structures. Surface plasmon resonance was also used to compute thenbinding while fluorescence resonance energy transfer-based assay was additionally used to confirm thenselectivity.Moreover, using real time PCRwe observed up to 90%downregulation of c-MYC transcripts aftern24 h of ligand treatment in HeLa cells. Using a luciferase assay we show the downregulation of the proteinnlevels. Fluorescent-assisted cell sorter-based cell cycle analysis showed a prominent arrest of cells in the sub-nG1 stage upon treatment of ligands that leads toward apoptosis. Altogether, these experiments support thenhypothesis that the present molecules are effective in specifically binding and stabilizing quadruplexes andnprovide a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.
机译：芽孢杆菌硫醇（Cys-GlcN-苹果酸，BSH）最近在炭疽芽孢杆菌，金黄色葡萄球菌和其他几种革兰氏阳性细菌中缺乏谷胱甘肽和霉菌硫醇而被鉴定为一种新型的低分子重量硫醇。我们现在已经表征了炭疽芽孢杆菌中BSH生物合成途径的前两种酶，它们结合起来可以从UDP-rnGlcNAc和L-苹果酸生产R-D-氨基葡萄糖L-苹果酸（GlcN-苹果酸）。已经确定了GlcNAc-苹果酸中间体的结构，以及BaBshA糖基转移酶（fGlcNAc-苹果酸）和BaBshB脱乙酰基酶（fGlcN-苹果酸）的动力学参数。 BSH是仅有的两种天然产物，据报道含有丙二糖苷，BaBshA-UDP-苹果酸三元复合物的晶体结构在本研究中以3.3 A的分辨率测定，确定了对特异性识别BSH重要的几个活性位点相互作用L-苹果酸，而不是其他rnR-羟基酸，作为受体底物。与报道的GlcNAc-1-D-核肌醇3-磷酸合酶（MshA）载脂蛋白和三元复合形式的结构形成鲜明对比，在相应的BaBshA形式的结构中未观察到主要构象变化。如所预测的，缺少BshA糖基转移酶的炭疽芽孢杆菌的突变株不能产生BSH。该炭疽杆菌bshA基因座（BA1558）已在转座子位点杂交研究中鉴定为生长，孢子形成或发芽所必需的[Day，WA，Jr.，Rasmussen，SL，Carpenter，BM，Peterson，SN和Friedlander， AMrn（2007）细菌学杂志。 189，3296-3301]，表明BSH的生物合成可能代表了新型抗菌剂的开发目标，这种抗菌剂具有针对革兰氏阳性病原体（如rnB）的广谱活性。炭疽病。在芽孢杆菌中硫醇氧化还原缓冲和体内稳态中起作用的代谢物未被很好地理解，并且我们基于此和其他近期工作提出了一张综合图片，这些配体可以有效地诱导富含G的22-mer c-MYC DNA序列中的G-四链体结构，并且进一步稳定结构。用等温滴定量热法测量的平衡结合常数表明，配体n对四链体结构（K〜107nM-1n）具有高亲和力，而对双链体DNA无亲和力，这表明配体对G-四链体结构具有选择性。表面等离振子共振也被用来计算然后结合，而荧光共振能量转移为基础的分析则进一步证实了选择性。此外，通过实时PCR，我们观察到HeLa细胞中配体处理24小时后c-MYC转录本下调了90％。使用荧光素酶测定，我们显示了蛋白水平的下调。基于荧光辅助细胞分选仪的细胞周期分析显示，在处理配体后会导致亚nG1期细胞明显停滞，导致凋亡。总之，这些实验支持以下假设：本发明的分子在特异性结合和稳定四链体方面有效，并且提供了合适的支架以发展成靶向四链体的治疗剂。

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  • 来源
    《Biochemistry》 |2010年第38期|p.8398-8414|共17页
  • 作者

    Derek Parsonage Gerald L. Newton Robert C. Holder Bret D. Wallace Carleitta Paige Chris J. Hamilton Patricia C. Dos Santos Matthew R. Redinbo Sean D. Reid and Al Claiborne;

  • 作者单位

    §Center for Structural Biology, and ) Department of Microbiology and Immunology, Wake Forest University School of Medicine,Winston-Salem, North Carolina 27157,^Department of Chemistry and Department of Biochemistry and Biophysics, University ofNorth Carolina, Chapel Hill, North Carolina 27599-3290,@Department of Chemistry, Wake Forest University, Winston-Salem,North Carolina 27109,#School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, England, andrDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0314.OThese authors contributed equally to this work.bPresent address: Department of Natural Sciences,Virginia Union University, Richmond, VA 23220.;

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