COMPARATIVE-ANALYSIS OF THE 5 MAJOR ERWINIA-CHRYSANTHEMI PECTATE LYASES - ENZYME CHARACTERISTICS AND POTENTIAL INHIBITORS

In Erwinia chrysanthemi 3937, pectate lyase activity mainly results fr om the cumulative action of five major isoenzymes, PelA to Pelf, Compa rison 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 meth od, We analyzed some of the basic enzymatic properties of these five i soenzymes, PelA has a low specific activity compared to the other four enzymes. PelB and PelC have a high affinity for their substrate: abou t 10-fold higher than the enzymes of the PelA-D-E group, The optimum p H 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 acti vities, 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 Pe lC, Enzymes of the PelB-C group are more stable than those of the PelA -D-E group, Use of substrates presenting various degrees of methylatio n revealed that PelA, PelD, and Pelf are active only for very low leve ls of methylation, while PelB and PelC are more active on partially me thylated pectins (up to 22% for PelC and up to 45% for PelB), Pectate lyases have an absolute requirement for Ca2+ ions, For the five isoenz ymes, 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 dem onstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM.