Structural dissection of the CMP-pseudaminic acid synthetase, PseF

Tessa Keenan; Andrew R Cowan; Emily K P Flack; Natasha E Hatton; Abigail J Walklett; Gavin H Thomas; Glyn R Hemsworth; Martin A Fascione

doi:10.1016/j.str.2024.09.017

Structural dissection of the CMP-pseudaminic acid synthetase, PseF

Structure. 2024 Oct 7:S0969-2126(24)00386-1. doi: 10.1016/j.str.2024.09.017. Online ahead of print.

Authors

Tessa Keenan¹, Andrew R Cowan², Emily K P Flack³, Natasha E Hatton¹, Abigail J Walklett¹, Gavin H Thomas³, Glyn R Hemsworth⁴, Martin A Fascione⁵

Affiliations

¹ Department of Chemistry, University of York, York YO10 5DD, UK.
² Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
³ Department of Biology, University of York, York YO10 5DD, UK.
⁴ Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Electronic address: g.r.hemsworth@leeds.ac.uk.
⁵ Department of Chemistry, University of York, York YO10 5DD, UK. Electronic address: martin.fascione@york.ac.uk.

PMID: 39393361
DOI: 10.1016/j.str.2024.09.017

Abstract

Pseudaminic acid is a non-mammalian sugar found in the surface glycoconjugates of many bacteria, including several human pathogens, and is a virulence factor thought to facilitate immune evasion. The final step in the biosynthesis of the nucleotide activated form of the sugar, CMP-Pse5Ac7Ac is performed by a CMP-Pse5Ac7Ac synthetase (PseF). Here we present the biochemical and structural characterization of PseF from Aeromonas caviae (AcPseF), with AcPseF displaying metal-dependent activity over a broad pH and temperature range. Upon binding to CMP-Pse5Ac7Ac, AcPseF undergoes dynamic movements akin to other CMP-ulosonic acid synthetases. The enzyme clearly discriminates Pse5Ac7Ac from other ulosonic acids, through active site interactions with side-chain functional groups and by positioning the molecule in a hydrophobic pocket. Finally, we show that AcPseF binds the CMP-Pse5Ac7Ac side chain in the lowest energy conformation, a trend that we observed in the structures of other enzymes of this class.

Keywords: Aeromonas caviae; CMP-Pseudaminic acid; CMP-Pseudaminic acid synthetase; PseF; pseudaminic acid; tg conformation; ulosonic acid.