The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB☆
Section snippets
General
Competent E. coli BLR(DE3) pLysS cells were purchased from Novagen (Madison, WI). The plasmid based on pET15b (Novagen) containing the gene truB[3] was generously provided by J. Ofengand; this plasmid will be referred to as pΨ55. Oligonucleotides (OPC-purified grade) were purchased from The Great American Gene company (Ramona, CA), and a Robocycler Gradient 96 Thermocycler (Stratagene, La Jolla, CA) was used for the PCR component of the site-directed mutagenesis. Ni–NTA Superflow resin, QIAprep
Generation, expression, and isolation of TruB variants
The site-directed mutagenesis proceeded smoothly, and all alterations were confirmed by sequencing the entire gene. Host cells harboring the plasmids for overexpression of all TruB variants grew normally. The variants retained a wild-type fold as judged by near-UV CD spectra that are essentially identical to those of wild-type TruB (data not shown). The stability of the altered TruB under the standard storage conditions was similar to that of wild-type enzyme except for H43G TruB, which lost
Discussion
An Asp (Asp-48 in TruB) is the only amino acid residue conserved among all Ψ synthases [6], [7], [8], and it is catalytically essential in all five families of these enzymes [8], [26], [27], [28], [29]. Two mechanisms have been proposed for the Ψ synthases [30], [32], and both invoke the essential Asp as a nucleophile, either to form a Michael adduct with the uracil ring or an acylal adduct with the ribose ring (Fig. 2). As originally formulated, both mechanisms also included the participation
Conclusions
The behavior of TruB upon replacement of Asp-48 with its sulfinic acid analog leads us to conclude that Asp-48 accounts for the ascending limb of the pH profile of TruB, and the linear dependence of log kcat on pH for the TruB with a cysteine sulfinic acid residue at position 48 can be explained if Asp-48 serves both as a nucleophile and as a general base in the final deprotonation to generate Ψ (Fig. 2). The descending limb may result from a pH-induced conformational change, service of Asp-48
Acknowledgments
We thank Karen Gartley for metal analysis, Paul F. Fitzpatrick for helpful discussions, and Steven L. Swann, Vidhyashankar Ramamurthy, Nabil Nasser, and Casey Boczon for conducting preliminary experiments.
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This work was supported by the National Institutes of Health (GM59636 to E.G.M) and the National Science Foundation (DBI0096578 and CHE9634238 to M.V.J.).