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

Acta Crystallogr F Struct Biol Commun. 2018 Aug 1;74(Pt 8):496-505. doi: 10.1107/S2053230X18006842. Epub 2018 Aug 1.

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.

Keywords: 2-oxohistidine; Clostridium; Ruminiclostridium thermocellum; bio-inorganic; carbohydrates; endoglucanase; enzymes; metal ion; oligosaccharides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites / physiology
  • Cellulase / chemistry*
  • Cellulase / genetics
  • Cellulase / metabolism
  • Crystallization
  • Metals, Heavy / chemistry*
  • Metals, Heavy / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary

Substances

  • Bacterial Proteins
  • Metals, Heavy
  • Cellulase