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.
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.
Sivaraman
J
J
Department of Biochemistry, McGill University, H3G 1Y6, Montreal, Que., Canada.
Iannuzzi
Pietro
P
Cygler
Miroslaw
M
Matte
Allan
A
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry*; D019753:Intramolecular Lyases / Q000737:chemistry; D019751:Intramolecular Transferases; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D040681:Structural Homology, Protein
D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D012313:RNA; D006836:Hydro-Lyases; C115603:RluD protein, E coli; C022479:pseudouridylate synthetase; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D019753:Intramolecular Lyases