Structural studies of the unusual metal-ion site of the GH124 endoglucanase from Ruminiclostridium thermocellum.

Gideon J Davies,Alan Cartmell,Saioa Urresti,Paul H Walton,Feng Liu

doi:10.1107/s2053230x18006842

Abstract

The recent discovery of `lytic' polysaccharide monooxygenases, copper-dependent enzymes for biomass degradation, has provided new impetus for the analysis of unusual metal-ion sites in carbohydrate-active enzymes. In this context, the CAZY family GH124 endoglucanase from Ruminiclostridium thermocellum contains an unusual metal-ion site, which was originally modelled as a Ca2+ site but features aspartic acid, asparagine and two histidine imidazoles as coordinating residues, which are more consistent with a transition-metal binding environment. It was sought to analyse whether the GH124 metal-ion site might accommodate other metals. It is demonstrated through thermal unfolding experiments that this metal-ion site can accommodate a range of transition metals (Fe2+, Cu2+, Mn2+ and Ni2+), whilst the three-dimensional structure and mass spectrometry show that one of the histidines is partially covalently modified and is present as a 2-oxohistidine residue; a feature that is rarely observed but that is believed to be involved in an `off-switch' to transition-metal binding. Atomic resolution (<1.1 Å) complexes define the metal-ion site and also reveal the binding of an unusual fructosylated oligosaccharide, which was presumably present as a contaminant in the cellohexaose used for crystallization. Although it has not been possible to detect a biological role for the unusual metal-ion site, this work highlights the need to study some of the many metal-ion sites in carbohydrate-active enzymes that have long been overlooked or previously mis-assigned.

Highlights

One of the most important recent developments in our understanding of the degradation of plant, and other recalcitrant, polysaccharides has been the discovery of polysaccharide monooxygenases (LPMOs, sometimes termed PMOs)
The original publication highlighted its catalytic activity as an unusual inverting cellulose
For R. thermocellum, RtGH124A is not targeted to the multi-enzyme cellulosome complex of the organism but instead consists of a ‘dockerin’ domain which instead targets the enzyme to the cell surface of the bacterium, followed by a CAZY GH124 catalytic domain; RtGh124 is an unusual enzyme if its primary role is in cellulose degradation yet it is not part of the cellulose complex

Summary

Introduction

One of the most important recent developments in our understanding of the degradation of plant, and other recalcitrant, polysaccharides has been the discovery of (lytic) polysaccharide monooxygenases (LPMOs, sometimes termed PMOs). LPMOs are oxygenases that harness an unusual copper-containing ‘histidine-brace’ active centre to generate reactive oxygen species (from O2 or, under specific laboratory conditions, H2O2), leading to oxidative chain cleavage (reviewed, for example, in Horn et al, 2012; Lo Leggio et al, 2012; Span & Marletta, 2015; Walton & Davies, 2016). These enzymes are classified in the Carbohydrate-Active enZymes (CAZy) classification as ‘auxiliary enzyme’ (Levasseur et al, 2013) activities AA9, AA10, AA11, AA13, AA14 and AA15. A further finding of this latter study was that one of the coordinating residues in fungal AA9 enzymes is a covalently modified, methylated histidine (Quinlan et al, 2011; Fig. 1)

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