Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

Abstract

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.

Figure 1. Three-dimensional structure of A. niger endoPGII

The β-helix encompasses ten complete rungs (numbered 1–10). The amino acid residues associated with subsites −3 to +3 are reported in the scheme [28]. Asp¹⁸⁰, Asp²⁰¹ and Asp²⁰² are involved in catalysis. The residues at subsites −1 and +1 shown in the table are strictly conserved among fungal endoPGs [12]. The Figure was prepared with PyMOL (DeLanoScientific; http://www.pymol.org). Amino acids mentioned in the table forming the bottom panel are given in one-letter code.

Figure 2. Electropherograms of a sample containing a mixture of OGAs with DPs 1–8 (A) and of the reaction mixture after 30 min of incubation at 37 °C on 0.25% PGA (B)

Details are given in the Experimental section.

Figure 3. Progression profile curves for released products on incubation of PGA (0.25 g/l) with BcPeh28A at 20 °C over 10 min (A) and over 1.5 h (B)

Δ, DP 1; ◆, DP 2; ▲, DP 3; ■, DP 4; □, DP 5; +, DP 6; ×, DP 7; *, DP 8. Details are given in the Experimental section.

Figure 4. Structure-based amino acid sequence alignment of A. niger endoPGII (PDB code 1CZF) and BcPeh28A

The amino acid residues of A. niger endoPGII shown in Figure 1 are in boldface and those that are conserved in BcPeh28A are also underlined. Ser⁹¹ (A. niger endoPGII) and Ser¹⁰⁴ (BcPeh28A) are underlined. They belong to rung 2 and are responsible for the non-processive enzymatic behaviour. The asterisk (*), colon (:) and dot (.) symbols indicate identical residues, conserved substitutions and semi-conserved substitutions respectively.

Figure 5. Reaction scheme (A), progression curves (B) and a graphical view of binding modes (C)

(A) Reaction scheme constructed on the basis of the product progression curves reported in Figure 3(B). (B) Progression curves of OGAs with DPs of 1–6 derived from the experimental data (continuous lines) and reconstructed according to the kinetic model (broken lines). (C) A graphical view of the productive binding modes between the enzyme and the substrates (oligomers with a DP of 4, 5 and 6 respectively). The reducing end of OGAs is indicated by the symbol Ø, whereas an arrow indicates the cleavage sites. The percentage of occurrence of each binding mode and the associated S.D. values are also reported.

Figure 6. HPAEC-PAD analysis of the hydrolysis of the decagalacturonide before (A) and after (B) incubation for 5 min at 37 °C with BcPeh28A

Details are given in the Experimental section.

Figure 7. Partially methyl-esterified hexagalacturonides (A), and methylation patterns of the hexagalacturonides (B)

The reducing end is indicated by the symbol Ø.

Figure 8. Mass spectra of the products released after the enzymatic hydrolysis by BcPeh28A of compound 1 after 1 h of incubation at 37 °C (A and B) and of compound 3 after 20 min of incubation at 37 °C (C and D)

The molecular masses indicated by the arrows were assigned as follows: peak 1: m/z 1139.25 [M+Na]⁺, hexagalacturonide 1; peaks 1a and 3a: m/z 407.08 [M+Na] ⁺, monomethylated dimer; peak 1b: m/z=773.16 [M+Na]⁺, dimethylated tetramer; peak 3b: m/z 583.11 [M+Na]⁺, monomethylated trimer. Details are given in the Experimental section.

Figure 9. BcPeh28A binding modes of compounds 1 (panel 1) and 3 (panels 2 and 3)

The reducing end is indicated by the symbol Ø, whereas an arrow indicates the cleavage sites.