Shevchik Vladimir E, Hugouvieux-Cotte-Pattat Nicole
Unité de Microbiologie et Génétique, UMR CNRS-INSA-UCB 5122, 69622 Villeurbanne, France.
J Bacteriol. 2003 May;185(10):3091-100. doi: 10.1128/JB.185.10.3091-3100.2003.
Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.
菊欧文氏菌通过酶解植物细胞壁中的果胶，引发多种植物的软腐病。果胶是一种复杂的多糖。其主链由半乳糖醛酸残基构成，其中一些残基被甲基和/或乙酰基酯化修饰。酯酶对于去除这些修饰、进而促进多糖链的进一步降解是必需的。除了在菊欧文氏菌3937菌株中鉴定出的首个果胶乙酰酯酶PaeY外，我们还发现该细菌产生由基因paeX编码的第二种果胶乙酰酯酶。paeX开放阅读框编码一个34940 Da的322个残基的前体蛋白，包括一个21个氨基酸的信号肽。通过基因融合分析paeX转录情况，发现它受果胶分解代谢产物诱导，并受分解代谢物阻遏的影响。paeX的表达受阻遏物KdgR调控，KdgR控制果胶分解代谢的所有步骤；受阻遏物PecS调控，PecS控制大多数果胶酶基因；还受分解代谢调节蛋白调控，分解代谢调节蛋白是糖分解代谢的全局激活剂。paeX基因位于参与果胶寡聚物分解代谢和转运的基因簇中。在诱导条件下，编码寡聚半乳糖醛酸特异性孔蛋白的两个相邻基因kdgM和paeX都从kdgM近端的启动子作为一个操纵子进行转录，但在非诱导条件下，paeX的转录也可与kdgM的转录解偶联。PaeX与溶纤维丁酸弧菌木聚糖酶XynB的C末端结构域以及一些细菌酯酶同源。PaeX含有对应于丝氨酸水解酶大家族活性位点的典型盒式结构（GxSxG）。纯化的PaeX能从各种合成底物和甜菜果胶中释放出乙酸盐。果胶经过先前的解聚和去甲基化后，PaeX的活性增加，这表明其优选的底物是未甲基化的寡聚半乳糖醛酸。PaeX主要存在于菊欧文氏菌的周质空间中。这些数据表明，PaeX主要参与通过KdgM孔蛋白进入周质的酯化寡聚半乳糖醛酸的脱乙酰化过程。
