Dissecting the Roles of a Strictly Conserved Tyrosine in Substrate Recognition and Catalysis by Pseudouridine 55 Synthase

Abstract

Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.

Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

“…The same is true for mutations at other positions (Y173 and R267) in hPus1p; some mutations result in no activity, such as Y173S, but others show some activity toward modification at position 27 in the S. cerevisiae pre-tRNA Ile . When the corresponding tyrosine in E. coli TruB (Y67) was mutated to phenylalanine, leucine, or alanine, there was no detectable activity on a natural tRNA substrate (Phannachet et al 2005), suggesting that there was a need for both a hydrophobic ring and an OH group combined in one side chain (Phannachet et al 2005). These data would suggest that the functions of these highly conserved amino acids are less constrained in hPus1p than for instance R62 in RluA, R58 in TruA, and Y67 in TruB.…”

“…Analysis of the TruA crystal structure in addition to structural studies of members of other C families has suggested that the two arginines (R58 and R205 in TruA) play a role in constraining the aspartate and in flipping out the uracil base of the substrate allowing it to be modified (Foster et al 2000;Hamma and Ferré-D'Amaré 2006;Hur et al 2006;Hur and Stroud 2007). The role of the tyrosine at 118 in TruA has not been fully elucidated; however, a previous mutation study using TruB C synthase has shown that replacement of the tyrosine with alanine, phenylalanine, or leucine results in the lack of enzymatic activity on a natural substrate (Phannachet et al 2005).…”

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

“…1B; Nurse et al 1995). TruB was the first pseudouridine synthase to be crystallized in complex with its substrate RNA (Hoang and Ferré-D'Amaré 2001), and numerous biochemical investigations have been performed to investigate its substrate specificity and catalytic mechanism Hamilton et al 2005;Hoang et al 2005;Phannachet et al 2005). Furthermore, TruB is of general interest as it is a close homolog and structurally very similar to the eukaryotic pseudouridine synthase Cbf5, the catalytic subunit of H/ACA small nucleolar ribonucleoproteins (Koonin 1996).…”

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

“…Pus10 does not contain this His. A Tyr residue in TruB that corresponds to Tyr339 of MjPus10, when mutated to Ala, Leu, or Phe, lost all of its Ψ55 synthase activity (Phannachet et al 2005). It has been proposed that this Tyr in TruB maintains the hydrophobic nature of the active site and its OH group is involved in catalysis (Phannachet et al 2005).…”

“…A Tyr residue in TruB that corresponds to Tyr339 of MjPus10, when mutated to Ala, Leu, or Phe, lost all of its Ψ55 synthase activity (Phannachet et al 2005). It has been proposed that this Tyr in TruB maintains the hydrophobic nature of the active site and its OH group is involved in catalysis (Phannachet et al 2005). Mutations of the corresponding Tyr in human PUS1 (member of TruA family) also show significant reduction of activity (Sibert et al 2008).…”