Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila.

Marco A S Kadowaki,Vincent G H Eijsink,Rolf A Prade,Anikó Várnai,Igor Polikarpov,John-Kristian Jameson,Patricia S Kumagai,Antonio J Costa-Filho,Ana E T Leite,Jean-Guy Berrin

doi:10.1371/journal.pone.0202148

Abstract

Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.

Highlights

Lytic Polysaccharide Monooxygenases (LPMOs) are oxidative enzymes able to boost the hydrolytic efficiency of glycoside hydrolases (GHs) during the depolymerization of recalcitrant polysaccharides [1, 2]
Phylogenetic analysis places MtLPMO9J in a cluster together with C4-oxidizing LPMOs; this cluster is divided into two groups (Fig 1A)
The group with MtLPMO9J consists of LPMOs (NcLPMO9C and PaLPMO9H) that are active on cello-oligosaccharides; the other group contains MtLPMO9C (MYCTH_100518) [13], which is not active on cello-oligosaccharides, and NcLPMO9A (NCU02240) and NcLPMO9D (NCU01050), LPMOs that accumulate C4-oxidized cellopentaose during their action on phosphoric acid-swollen cellulose (PASC) [19] and, are likely not active on cello-oligosaccharides

Summary

Introduction

Lytic Polysaccharide Monooxygenases (LPMOs) are oxidative enzymes able to boost the hydrolytic efficiency of glycoside hydrolases (GHs) during the depolymerization of recalcitrant polysaccharides (such as lignocellulose and chitin) [1, 2]. The contribution of additional factors [4] and redox enzymes [5] has been predicted since 1950, the existence and structure of LPMOs have been described just recently [2, 6,7,8], and their mechanism is still under debate [9,10,11]. LPMOs are metalloenzymes, with a Cu(I/II) ion coordinated by two histidines forming a His-brace in the active site [2, 6]. In fungal LPMOs, one of the coordinating histidines, the N-terminal His residue (His1), is methylated at Nε2 [6]. LPMO action requires an external electron donor [2] and molecular oxygen [2] or hydrogen peroxide [10]. The attack of LPMOs on the surface of crystalline cellulose disrupts the crystalline structure and introduces new binding sites for cellulases [20, 21]

Methods

Results

Conclusion