Site-specific RluE makes pseudouridine at position 2457 in a short stem region of helix 89 of Domain V in 23S RNA. The protein has an active site cleft, conserved in all other pseudouridine synthases, that contains invariant Asp and Tyr residues implicated in catalysis. A positively charged surface patch that covers the active site cleft is just wide enough to accommodate an RNA stem. The RNA substrate stem can be docked to this surface such that the catalytic Asp is adjacent to the target base, and a conserved Arg is positioned to help flip the target base out of the stem into the enzyme active site. However the stem alone is not a good RluE substrate, suggesting RluE makes additional interactions with other regions in the ribosome. A flexible RluE loop lies close to the conserved region of the stem in the model and may contribute to substrate specificity. RluE belongs to the same subgroup of RNA pseudouridine synthases as RsuA, RluB and RluF.