The Study Of Aminoacyl-tRNA Synthetase/Protein Engineer Of Glutaryl 7-amino-cephalosporin Acid Acylase

Arginyl-tRNA synthetase (ArgRS) is one of components of a macromolecularcomplex consisting of at least nine tRNA synthetases and three auxiliary proteinsfrom Drosophila to human. In mammalian cells, ArgRS is present as a free protein aswell as a component of the complex. The ArgRS in the complex (cArgRS) has apeptide extension consisting of 72 amino acids appended to it N-terminus comparedwith the free ArgRS (?NArgRS). We found that there is an up-stream AUG (uAUG)codon before the two AUG codons in a same open-reading frame (ORF)corresponding to the two forms of ArgRS respectively. Our results demonstrated thattwo forms of human cytoplasmic ArgRSs could be produced by alternative translationinitiation from the two AUG codons in the same ORF. And the uAUG can increasethe production of the free ArgRS. The kinetics of inhibition of E. coli, yeast and human ArgRS by L-canavaninewere analyzed. The inhibition constant (Ki) for the three ArgRS was 15μM (E. coli),90μM (yeast) and 1.5mM (human), respectively. A three-dimension structure modelof human ArgRS was constructed on the basis of the crystal structure of yeast ArgRS.Model caparison suggested that there should be difference between the structure ofthe L-arginine binding site of human ArgRS and the E. coli ArgRS. And the structure -5-中国科学院生物化学与细胞生物学研究所博士论文 郑勇刚 摘要difference should be responsible to the inhibition difference of the two ArgRSs by L-canavanine. L-canavanine has potency to be developed to a novel anti-bacteria drug. The Aquifex aeolicus αβ-LeuRS is the only known heterodimeric class Iaaminoacyl-tRNA synthetase. In this study, we investigated the function of the βsubunit which is believed to bind tRNALeu. A yeast three-hybrid system wasconstructed on the basis of the interaction of the β subunit with its cognate tRNALeu.Then, seven mutated β subunits exhibiting impaired tRNA binding capacities wereselected out from a randomly mutated library. Two mutations were identified in theclass Ia-helix-bundle domain, which might interact with the D-hairpin of the tRNAanalogous to other class Ia tRNA:synthetases complexes. The five other mutationswere found in the LeuRS-specific carboxy-terminal domain of which the folding isstill unknown. tRNA affinity measurements and kinetic analyses performed on theisolated β subunits and on the co-expressed αβ-heterodimers showed for all themutants an effect in tRNA affinity in the ground state. In addition, an effect on thetransition state of the aminoacylation reaction was observed for a 21-residues deletionmutant of the carboxy-terminal end. These results show that the genetic approach ofthe three hybrid system is widely applicable and is a powerful tool for theinvestigation of tRNA:synthetase interactions. We have identified a series of mutants of the β-subunit of A. aeolicus αβ-LeuRS with tRNA binding capacity reduced but found that only one mutant N152Aimpaired the transition state of aminoacylation of αβ-LeuRS. Asn152 is an invariableresidue in all reported LeuRS. 3D structure analysis demonstrated that Asn152 couldform a water molecule-mediated hydrogen bond with another invariable residue Pro77. -6-中国科学院生物化学与细胞生物学研究所博士论文 郑勇刚 摘要Mutational substitute of Pro77 also affected the transition state of aminoacylation.And the double mutants P77G/N152A presented the similar kinetics constants withthe two single mutants N152A and P77G that suggested the two residues are bothrequired performing the function. Mutation of the two residues has no effects on theleucine minihelix recognition by αβ-LeuRS. But the mutated enzymes lost the abilityto discriminate minihelix with a small...