# Structural characterisation of nucleotide sugar short‐chain dehydrogenases/reductases from the thermophilic pseudomurein‐containing methanogen Methanothermobacter thermautotrophicus ΔH

## TL;DR

The paper describes the structural analysis of nucleotide sugar enzymes in a methanogen, revealing their roles in pseudomurein and polysaccharide biosynthesis.

## Contribution

The study provides novel structural and functional insights into archaeal nucleotide sugar enzymes involved in glycan biosynthesis.

## Key findings

- Mth375 structure reveals a Rossmann-fold domain with NADH and UDP binding.
- Mth373 is identified as a UDP-xylose synthase with a 1.96 Å resolution structure.
- Molecular modeling identifies Mth380, Mth631, and Mth1789 as specific nucleotide sugar enzymes.

## Abstract

Epimerases and dehydratases are widely studied members of the extended short‐chain dehydrogenase/reductase (SDR) enzyme superfamily and are important in nucleotide sugar conversion and diversification, for example, the interconversion of uridine diphosphate (UDP)‐linked glucose and galactose. Methanothermobacter thermautotrophicus contains a cluster of genes, the annotations of which indicate involvement in glycan biosynthesis such as that of cell walls or capsular polysaccharides. In particular, genes encoding UDP‐glucose 4‐epimerase related protein (Mth375), UDP‐glucose 4‐epimerase homologue (Mth380) and dTDP‐glucose 4,6‐dehydratase related protein (Mth373) may be involved in the biosynthesis of an unusual aminosugar in pseudomurein. In this paper, we present the structures of Mth375, an archaeal sugar epimerase/dehydratase protein (WbmF) determined to a resolution of 2.0 Å. The structure contains an N‐terminal Rossmann‐fold domain with bound nicotinamide adenine dinucleotide hydride (NADH) and a C‐terminal catalytic domain with bound UDP. We also present the structure for Mth373 co‐crystallised with uridine‐5′‐diphosphate‐xylopyranose to a resolution of 1.96 Å as a NAD+‐dependent oxidative decarboxylase (UDP‐xylose synthase; EC4.1.1.35). Molecular modelling has also allowed for the identification of Mth380 as a UDP‐N‐acetylglucosamine 4‐epimerase (WbpP; EC5.1.3.7), Mth631 as a UDP‐glucose 4‐epimerase (GalE; EC5.1.3.2) and Mth1789 as a classical dTDP‐d‐glucose 4,6‐dehydratase (EC4.2.1.46). The UDP–sugar specificity of each archaeal nucleotide sugar short‐chain dehydrogenase/reductase (NS‐SDR) was elucidated via sequence, molecular modelling and structural analyses. Overall, these structures potentially shed light on the formation of the glycan portion of pseudomurein and capsular polysaccharide in Archaea.

Nucleotide sugar short‐chain dehydrogenases/reductases (NS‐SDRs) are involved in pseudomurein and capsular polysaccharide formation in methanogenic Archaea. Methanothermobacter thermautotrophicus possesses several NS‐SDRs labelled Mth375, Mth380, Mth373, Mth631 and Mth1789. Utilising X‐ray crystallography, molecular modelling and phylogenetics we shed light on the NS‐SDRs of M. thermautotrophicus and their potential enzyme activities.