Pectin and pectate lyases

PL 1 pectate lyases are Ca dependent, but pectin lyases are not, although some stimulation of activity at high pFl is observed the pH optimum of pectate lyases is about 8.5 and that of pectin lyases about 5.5. Comparison of the structures of the PL 1 pectin lyase A and from Aspergillus niger with PL 1 pectate lyases reveals that a Ca -binding carboxylate is replaced by an arginine. The probable 10 units difference in the values of C5-H5 in the 

The Family 3 structure is similar in structure to Family 1 pectate lyases, the pectate lyase of a Bacillus species being a parallel eight-turn (3-helix.Site-directed mutagenesis experiments supported a role for an arginine as the active site base that deprotonated C5, with a role for the second Ca in stabilising the enolate (the first Ca being structural). It is possible that the very existence of a Family 3 PL, separate from Family 1, is an artefact of the many basic residues incorporated into this enzyme that ensure its alkaline pFl optimum. 

The Family 9 pectate lyase from the plant pathogen Erwinia chrysanthemi has again a structure composed largely of (3-strands, being a 10-turn helix of parallel (3-strands. Analogy with the closely similar PL 1 enzymes suggests that the enzyme binds a catalytic Ca between enzyme and substrate, in addition to 

No dedicated acid catalyst which donates a proton to 04 has as yet been found in any pectin or pectate lyase transfer from solvent or from more remote protein acids and bases via a Grotthus mechanism appears to be all that is required. 

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The distribution of methoxyl groups in apple and citrus pectic substances13711 has been assessed by fractionation of degradation products released by pectin- and pectate-lyases. 

Endo-PL was found to be inactive on pectate, pectic acid amide and glycolester of pectate endo-PALs have similar activity on glycolesters of pectate as on the methylesters. Activity on glycolesters of pectate was therefore suggested as a criterion to distinguish between pectin and pectate lyases (27,34). 

Figure 6.74 Proposed mechanism of pectin and pectate lyases, (a) Pectin lyases, (b) Pectate lyases in PL the base is a lysine. Note that detailed mechanistic investigations, similar to PL 8, are lacking.

Pectin and pectate lyases are only produced by microorganisms. They are activated by Ca " ions and differ in the pH optimum (pH 8.5-9.5) from the polygalacturonases (pH 5-6.5). 

Pectin lyase and pectate lyase activities were assayed by measuring the increasing of the absorbancy at 235 nm by the method of Albersheim and Killias (12, 13) when pectin or sodium pectate was used as the substrate, repectively. 

The PNL exhibits an optimum pH of 9.5 (figure 6) and an optimum temperature of 55" C (figure 7). Lyases catalyze the reaction in an alkaline or in a neutral medium at high temperatures [32] pectin lyase from Phoma medicaginis var. pinodella showed an optimum pH of 7.5 [25], endopectate lyase from Fusarium solani f sp. pisi showed an optimum pH of 9.4 [31], and pectate lyase fi om Rhizoctonia solani showed an optimum pH of 8.0 [34]. 

Most commercial products are a mixture essentially of four individual enzymes pectin esterases, polygalacturonases, pectin lyases and pectate lyases. Depending on the intended use, the enzyme preparations have different contents of the individual pectolytic enzymes. The enzyme preparations are commercially available in liquid or solid form they originate mainly from mould cultures. 

Figure 8. High-pressure liquid chromatograms of pectin fractions degraded with pectin lyase (a) and pectate lyase (b) to degradation limits, respectively, of 18< and 11>. (Reproduced with permission from reference 4. Copyright 1983 Carbohydrate Polymer).

Optimal conditions for the synthesis of pectin lyase by Rhizoctania solani have been sought, and pectate lyase has been found in Sphaeropsis malocum growing on apple tissues. ... 

Bartling, S., Wegener, C. and Olsen, O. (1995). Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin. Microbiology 141,873-881. 

Erwinia chrysanthemi synthesizes and secretes a large number of pectinases. The major pectinases include a pectin methylesterase PemA and five isoenzymes of endo-pectate lyases PelA, PelB, PelC, PelD and PelE. In addition, secondary pectinases were identified a pectin methylesterase PemB, two endo-pectate lyases PelL and PelZ, an exo-pectate lyase PelX and an exopolygalacturonase, PehX. The regulation of pectinase synthesis is very complex and dependent on many environmental conditions. It is induced by pectin catabolic products and affected by growth phase, catabolite repression, osmolarity, iron or oxygen starvation... 

Structural analysis of the two pectate lyases PelC and PelE (5, 6), demonstrated that these proteins fold in a large heHx of parallel P strands. A stack of asparagine residues parallel to the helix probably plays a role in the stabUity of this structure. Identification of the structurally conserved amino adds lead to a reaHgnment of the protein sequences (7). In addition to Erwinia extracellular pectate lyases, the multiple aHgnment indudes the Bacillus subtilis pectate lyase, Aspergillus tdger and E. carotovora pectin lyases and plant proteins. 

Digestion of PGA by the PelL enzyme yielded a mixture of unsaturated ohgogalacturonides, giving evidence that PelL is an endo-deaving lyase (17). An exo-enz3mie, such as the EC 16 PelX, would generate a single product (15). The PelL protein differs from the major E. chrysanthemi pectate lyases in its ability to cleave both PGA and methylated pectin (17). The PelL activity has a basic optimum pH and an absolute requirement for Ca + ions. Analysis of culture supernatants demonstrated that PelL is an extracellular enzyme, such as the other secondary pectate lyases (17). 

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