STRUCTURAL DETERMINATION OF QUEA ENZYMES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The QueA enzyme catalyzes the formation of the 2,3-epoxy-4,5-dihydroxycyclopentane ring of the Q precursor epoxyqueuosine (oQ). S-adenosyl-L-methionine (AdoMet) reacts with 7-aminomethyl-7-deazaguanine of tRNA at position 34 to yield adenine, methionine, and a modified tRNA with oQ at position 34. The epoxy-cyclopentenediol moiety of oQ originates from the ribosyl portion of AdoMet, which is the only known example of the stoichiometric use of AdoMet as a ribosyl donor in an enzymatic reaction. Queuosine {7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)-methyl)-7-deazaguanosine} is a hypermodified nucleoside that occurs at position 34, the anticodon wobble position, of aspartate, asparagine, tyrosine, and histidine tRNAs. Although queuosine is found in bacteria and eukaryotes, it is synthesized de novo exclusively in bacteria. Therefore, eukaryotes obtain queuosine as a dietary nutrient and from intestinal flora, and incorporate the corresponding base queuine into tRNA using the enzyme queuine-tRNA ribosyltransferase. We have started crystallization attempts on the wild type enzyme and a GST (Glutathione-s-transferase)-fused enzyme. The formation of fusion proteins containing the desired peptide or protein of interest have been successfully used for the crystallization of difficult proteins. Because of the initial difficulty we had with the crystallization of the wild type enzyme, we started parallel crystallization attempt of QueA as a GST-fusion protein. It is also important to note that the GST-fusion protein showed similar activity to the wild type QueA enzyme. This would mean that the active site and the RNA binding site are not blocked by the GST tag. Therefore, we are also attempting the crystallization of the complexes of the QueA:GST protein with substrate and cofactor. The initial results show that the GST-fusion protein is a better candidate for crystallization. We have small crystals of the QueA-GST-fusion protein which showed diffraction to around 3.0 ¿ resolution.