A phospho-sugar binding domain homologous to NagB enzymes regulates the activity of the central glycolytic genes repressor
A phospho-sugar binding domain homologous to NagB enzymes regulates the activity of the central glycolytic genes repressor
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DOI：
10.1002/prot.21883
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发表时间：
2008-06-01
影响因子：
2.9
通讯作者：
Declerck, Nathalie
中科院分区：
文献类型：
--
作者：
Doan, Thierry;Martin, Laetitia;Declerck, Nathalie
CggR属于细菌转录调节因子的SorC家族，其控制参与碳水化合物催化的基因和操纵子的表达。CggR首先在枯草芽孢杆菌中鉴定，其中它抑制编码催化糖酵解的中心部分的五种酶的gapA操纵子。在这里，我们提出了一个结构/功能的研究表明，CggR的C-末端区域调节的DNA结合活性的阻遏物在响应结合的磷酸化糖。CggR的分子模型揭示了一个翼螺旋DNA结合基序，随后是一个C-末端结构域，与来自NagB家族的葡萄糖胺-6-磷酸脱氨酶具有弱但显著的同源性。计算机配体筛选表明，CggR C-末端结构域将优先结合双磷酸化的化合物，与先前提出的果糖-1，6-二磷酸（FBP）作为诱导剂代谢物的研究一致。在体外，FBP是唯一能够干扰CggR与DNA的协同结合的糖化合物。还发现FBP保护CggR免受胰蛋白酶降解，预测两个精氨酸残基位于形成NagB酶活性位点lid的移动的环中。预测与FBP相互作用的残基的替换导致突变体CggR在体内具有改变的阻遏物活性，但在体外保留其结构完整性和DNA结合活性。有趣的是，一些突变阻遏物对单-和二-磷酸-果糖苷具有不同的特异性。基于这些结果，我们提出，CggR样阻遏物的活性是由一个磷酸糖结合（PSB）结构域与NagB酶的结构和功能同源性控制。
CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes.