Shevchik V E, Hugouvieux-Cotte-Pattat N
Laboratoire de Genetique Moleculaire des Microorganismes, CNRS-UMR 5577, INSA, Villeurbanne, France.
Mol Microbiol. 1997 Jun;24(6):1285-301. doi: 10.1046/j.1365-2958.1997.4331800.x.
Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease.
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。果胶的结构复杂性要求多种果胶酶共同作用才能有效分解。到目前为止，在菊欧文氏菌中已鉴定出三种类型的果胶酶：两种果胶甲基酯酶（PemA、PemB）、一种聚半乳糖醛酸酶（PehX）和八种果胶酸裂解酶（PelA、PelB、PelC、PelD、PelE、PelL、PelZ、PelX）。我们在本文中报道了对一种新型酶——由paeY基因编码的果胶乙酰酯酶的分析。此前尚未描述过细菌形式的果胶乙酰酯酶，而植物组织和一些果胶分解真菌被发现能产生类似的酶。paeY基因存在于一个由五个果胶酶编码基因组成的簇中，即pelA - pelE - pelD - paeY - pemA。paeY开放阅读框长1650个碱基，编码一个60704Da的551个氨基酸的前体蛋白，包括一个25个氨基酸的信号肽。PaeY与棘孢曲霉的鼠李半乳糖醛酸乙酰酯酶有一个同源区域。为了表征该酶，paeY基因被过量表达，其蛋白质产物被纯化。PaeY能从甜菜果胶和各种合成底物中释放乙酸。此外，该酶与其他果胶酶协同作用。在果胶经PemA预先脱甲基以及经果胶酸裂解酶解聚后，PaeY的脱酯率增加。此外，在分别经PemA和PaeY去除甲基和乙酰基后，果胶酸裂解酶对甜菜果胶的降解更有利。paeY基因最初是根据其调控被鉴定出来的，它与其他果胶酶的调控有几个共同特征。利用基因融合对paeY转录进行分析表明，它受果胶分解代谢产物诱导，受生长阶段、氧限制和分解代谢物阻遏的影响。paeY表达的调控似乎依赖于控制果胶分解代谢所有步骤的KdgR阻遏物以及糖分解代谢的全局激活剂——分解代谢调节蛋白（CRP）。相邻的pelD、paeY和pemA基因从pelD近端的启动子转录为一个操纵子，这使得它们能受KdgR和CRP调控。然而，转录可在位于pelD和paeY之间的操纵子内不依赖Rho的终止子处中断。接种到非洲紫罗兰植株中的paeY突变体的侵染性低于野生型菊欧文氏菌菌株3937，这表明PaeY在软腐病中起重要作用。
