MUTAGENESIS AND CRYSTALLOGRAPHIC STUDIES OF ZYMOMONAS-MOBILIS TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE REVEAL ASPARTATE 102 AS THE ACTIVE-SITENUCLEOPHILE

Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttran scriptional base exchange of the queuine precursor 7-aminomethyl-7-dea zaguanine (preQ(1)) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray stru ctures of Zymomonas mobilis TGT and of its complex with preQ(1) [Romie r, C., Reuter, K,, Suck, D., Br Ficner, R. (1996) EMBO J. 15, 2850-285 7] have revealed a specific preQ(1) binding pocket and allowed a propo sal : for tRNA binding and recognition, We have used band-shift experi ments in denaturing conditions to study the enzymatic reaction perform ed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate, Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation o f this residue into alanine by site-directed mutagenesis leads to an i nactive mutant unable to form a covalent intermediate with tRNA, provi ng that aspartate 102 is the active site nucleophile in TGT. To invest igate the recognition of the wobble guanine in the preQ(1) binding poc ket, we mutated aspartate 156, the major recognition element for preQ( 1), into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent i ntermediate with tRNA in the first step of the reaction mechanism in c omparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ(1) binding pocket is required for the cleav age of the glycosidic bond, The three mutants were crystallized and th eir X-ray structures determined. The mutants display only subtle chang es to the wildtype protein, confirming that the observed biochemical r esults are due to the chemical substitutions rather than structural re arrangements.