The Crystal Structure of dTDP-D-glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-L-rhamnose pathway

STM Allard, MF Giraud, C Whitfield, M Graninger, P Messner, James Henderson Naismith

Research output: Contribution to journalArticlepeer-review

115 Citations (Scopus)

Abstract

(L)-Rhamnose is a 6-deoxyhexose that is found in a variety of different glycoconjugates in the cell walls of pathogenic bacteria. The precursor of L-rhamnose is dTDP-(L)-rhamnose, which is synthesised from glucose-1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway requiring four enzymes. Significantly this pathway does not exist in humans and all four enzymes therefore represent potential therapeutic targets, dTDP-(D)-glucose 4,6-dehydratase (Rm1B; EC 4.2.1.46) is the second enzyme in the dTDP-(L)-rhamnose biosynthetic pathway. The structure of Salmonella enterica serovar Typhimurium Rm1B had been determined to 2.47 Angstrom resolution with its cofactor NAD (+) bound. The structure has been refined to a crystallographic X-factor of 20.4% and an X-free value of 24.9 % with good stereochemistry.

Rm1B functions as a homodimer with monomer association occurring principally through hydrophobic interactions via a four-helix bundle. Each monomer exhibits an alpha/beta structure that can be divided into two domains. The larger N-terminal domain binds the nucleotide cofactor NAD + and consists of a seven-stranded P-sheet surrounded by alpha -helices. The smaller C-terminal domain is responsible for binding the sugar substrate dTDP-(D)-glucose and contains four beta -strands and six alpha -helices. The two domains meet to form a cavity in the enzyme. The highly conserved active site Tyr(167)XXXLys(171) catalytic couple and the GlyXGlyXXGly motif at the N terminus characterise Rm1B as a member of the short-chain dehydrogenase/reductase extended family.

The quaternary structure of Rm1B and its similarity to a number of other closely related short-chain dehydrogenase/reductase enzymes have enabled us to propose a mechanism of catalysis for this important enzyme. (C) 2001 Academic Press.

Original languageEnglish
Pages (from-to)283-295
Number of pages13
JournalJournal of Molecular Biology
Volume307
DOIs
Publication statusPublished - 16 Mar 2001

Keywords

  • rhamnose
  • RmlB
  • dTDP-D-glucose 4,6-dehydratase
  • crystal structure
  • Salmonella enterica serovar Typhimurium
  • UDP-GALACTOSE 4-EPIMERASE
  • ELECTRON-DENSITY MAPS
  • ESCHERICHIA-COLI
  • STREPTOCOCCUS-SUIS
  • CATALYTIC MECHANISM
  • DEOXY SUGARS
  • GENE-CLUSTER
  • CHAIN-LENGTH
  • IDENTIFICATION
  • VIRULENCE

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