Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.

Julian Quehenberger,Lukas Rettenbacher,Niklas Baumann,Oliver Spadiut,Tom Reichenbach,Christina Divne

doi:10.3390/ijms20010185

Julian Quehenberger, Lukas Rettenbacher + Show 4 more

Open Access

https://doi.org/10.3390/ijms20010185

Copy DOI

Abstract

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Highlights

Chaetomium thermophilum is a thermophilic fungus of the phylum Ascomycota
191 Protein Data Bank (PDB) entries for C. thermophilum have been deposited to this date; a number that has been rapidly increasing in recent years compared to only 19 entries until the year 2013 [1]
The xylose reductase (XR) gene was identified in the genome of C. thermophilum DSM 1495 by homology search and the construct was ordered from genscript (Piscataway, NJ, USA), codon optimized for Escherichia coli

Summary

Introduction

Chaetomium thermophilum is a thermophilic fungus of the phylum Ascomycota It is one of the most thermophile Eukaryotes, growing optimally at temperatures of 50–55 °C on rotting organic matter [1]. The complete nuclear and mitochondrial genomes of C. thermophilum are sequenced and the nuclear genome is estimated to be 28.3 Mbp with 7227 open reading frames while the mitochondrial genome is 127 kbp with 15 protein coding genes [2]. From an industrial point of view the organism is especially interesting for prospecting thermostable enzymes that are exclusively present in Eukaryotes. These enzymes cannot be found in extreme thermophiles (growth at 85 °C), a group of organisms that are only found in the prokaryotic Domains Bacteria and Archaea [3]

Methods

Results

Conclusion