Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

J Mol Biol. 2004 Jan 2;335(1):87-101. doi: 10.1016/j.jmb.2003.10.003.

Abstract

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray*
  • Escherichia coli / enzymology
  • Escherichia coli / growth & development
  • Escherichia coli Proteins / chemistry*
  • Hydro-Lyases / chemistry*
  • Intramolecular Lyases / chemistry
  • Intramolecular Transferases
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA / chemistry
  • RNA, Ribosomal, 23S / metabolism
  • Structural Homology, Protein

Substances

  • Escherichia coli Proteins
  • RNA, Ribosomal, 23S
  • RNA
  • Hydro-Lyases
  • RluD protein, E coli
  • pseudouridylate synthetase
  • Intramolecular Transferases
  • pseudouridine synthases
  • Intramolecular Lyases

Associated data

  • PDB/1PRZ