Side-by-side biochemical comparison of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare on their activity against crystalline cellulose and glucomannan

Bing Liu,Jerry Ståhlberg,Sumitha Krishnaswamyreddy,Anders Broberg,Mats Sandgren,Åke Olson,Madhu Nair Muraleedharan,Daniel Cullen

doi:10.1371/journal.pone.0203430

Abstract

Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLPMO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.

Highlights

Fungal colonization of wood is associated with decomposition of plant biomass to sustain the growing mycelia
In this study we present a side-by-side comparison of HiLPMO9B and HiLPMO9I in terms of activity on bacterial microcrystalline cellulose (BMCC) and cooperation with the H. irregulare glycoside hydrolase family 7 cellobiohydrolase HiCel7A, as well as their glucomannan activity and detailed analysis of glucomannan products generated by HiLPMO9I and structural factors of HiLPMO9I important for glucomannan binding
Bacterial microcrystalline cellulose at 1 mg/ml concentration was incubated for 48 h at 20 ̊C, pH 5.0, without and with the addition of 0.5 μM of HiLPMO9B or HiLPMO9I, in the presence of 1mM pyrogallol as reducing agent; thereafter the residual cellulose was washed to remove soluble sugars

Summary

Introduction

Fungal colonization of wood is associated with decomposition of plant biomass to sustain the growing mycelia. H. irregulare was grown on woody biomass, and the most abundant protein by far in the culture filtrate was identified as cellobiohydrolase HiCel7A [17] It is the only family 7 glycoside hydrolase in the genome of H. irregulare and it consists of a single catalytic domain without attached linker region and carbohydrate-binding module (CBM). Two H. irregulare LPMOs differ activity in degradation of crystalline cellulose and glucomannan different polysaccharide components in plant cell walls [18,26,32,33,34,35,36,37]. In this study we present a side-by-side comparison of HiLPMO9B and HiLPMO9I in terms of activity on bacterial microcrystalline cellulose (BMCC) and cooperation with the H. irregulare glycoside hydrolase family 7 cellobiohydrolase HiCel7A, as well as their glucomannan activity and detailed analysis of glucomannan products generated by HiLPMO9I and structural factors of HiLPMO9I important for glucomannan binding

Materials and methods

Results

Discussion