Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: Enzyme characteristics and potential inhibitors
Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: Enzyme characteristics and potential inhibitors
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DOI：
10.1128/jb.179.8.2503-2511.1997
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发表时间：
1997-04-01
影响因子：
3.2
通讯作者：
HugouvieuxCottePattat, N
中科院分区：
文献类型：
--
作者：
Tardy, F;Nasser, W;HugouvieuxCottePattat, N
在欧文氏菌3937中，果胶酸裂解酶活性主要是由PelA至Pelf五种主要同工酶的累积作用引起的。比较它们的氨基酸序列，发现有PelB-C和PelA-D-E两个家族。通过分子克隆技术在大肠杆菌中表达不同的pel基因，并分离出与其他同工酶不同的Pel基因。我们采用相似的实验条件，用一步层析法大量生产并纯化了五种pel基因。PelB和PelC对其底物具有高亲和力：比PelA-D-E组的酶高约10倍，PeLB和PelC的最适pH更碱性（约9.2）比PelA、PelD和Pelf（从8到8.8），在pH 7以下，Pelf和PelC的活性可以忽略不计，而PelA、PelD和Pelf保留了25%到30%的活性，对于PelD和Pelf，确定最适温度为50 ℃，对于PelA为55 ℃，对于PelB和PelC为60 ℃。PelB-C组的酶比PelA-D-E组的酶更稳定。使用呈现不同甲基化程度的底物显示PelA、PelD和Pelf仅对非常低水平的甲基化有活性，而PelB和PelC对部分甲基化的果胶更有活性，果胶酸裂解酶对Ca 2+离子具有绝对需求。对于五种同工酶，在0.1 mM的Ca 2+浓度下获得最大活性。（Ba ~（2+）、Co ~（2+）、Cu ~（2+）、Mg ~（2+）、Mn ~（2+）、Sr ~（2+）、Zn ~（2+））可替代Ca ~（2+）。在高浓度（1 mM）下，大多数二价阳离子抑制果胶酸裂解酶活性。此外，我们证明了存在于植物组织中的两种化合物，表儿茶素和水杨酸，在0.2mM的浓度下抑制果胶酸裂解酶。
In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to Pelf, Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E. Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes, We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method, We analyzed some of the basic enzymatic properties of these five isoenzymes, PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group, The optimum pH is more alkaline for PeLB and PelC (about 9.2) than for PelA, PelD, and Pelf (from 8 to 8.8), Below pH 7, activity was negligible for Pelf and PelC, while PelA, PelD, and Pelf retained 25 to 30% of their activities, The temperature optima were determined to be 50 degrees C for PelD and Pelf, 55 degrees C for PelA, and 60 degrees C for PelB and PelC, Enzymes of the PelB-C group are more stable than those of the PelA-D-E group, Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and Pelf are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB), Pectate lyases have an absolute requirement for Ca2+ ions, For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM. None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+. At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity, In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM.