Critical aspartic acid residues in pseudouridine synthases.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

Identification of a gene involved in the generation of 4-thiouridine in tRNA.

All organisms modify the bases of their RNA after transcription. Relatively little is known about the functions that these chemical alterations serve and, with very few exceptions, even less has been established regarding the enzymology involved. One modified base of known function is 4-thiouridine at position 8 of certain bacterial tRNAs, which serves as a photosensor for near-UV light. A gene involved in the conversion of uridine at position 8 into 4-thiouridine has been identified by genetic screening and its role in 4-thiouridine generation has been confirmed biochemically. This same gene, thiI , has recently been shown to play a role in thiamin biosynthesis. The purification and characteristics of the purified protein are also reported.