Study on the putative active site of Enterococcus faecalis prolyl- tRNA synthetase editing domain by methods of site-directed mutagenesis

Abstract

The maintenance of amino acid specificity by aminoacyl-tRNA synthetases can require the hydrolysis of missynthesized products that is known as amino acid editing. Bacterial prolyl-tRNA synthetase includes a special editing domain, that deacylates alanyl-tRNAPro, and so exhibits post-transfer editing activity. The mechanism of tRNA-dependent editing by prolyl-tRNA synthetase has to be defined. The present work aim is to study the structure of the active site of enterobacteria E. faecalis prolyl-tRNA synthetase editing domain. The amino acids positions E218, T257, K279, G331, S332, G334, and H366 have been chosen for the site-directed mutagenesis (alanine scanning). An editing activity of the mutants was compared with the wild type prolyl-tRNA synthetase. Three amino acid residues, important for the editing activity, K279, G331 and H366, were revealed. This data are consistent with the existing suppositions about the structure of bacterial prolyl-tRNA synthetase deacylating active sit

Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

“…The bacterial ProRS INS domain has been previously investigated using biochemical, 13,22,41 structural, 42 and computational studies. 22 Although there is no available crystal structure of an INS domain-tRNA complex, the computational model of E. faecalis INS bound to 5′-CCA-Ala, 22 revealed that the aminoacyl moiety binds in a well-defined hydrophobic pocket formed by residues Ile263, Val266, Ile277, and Lys279 and rationalizes the high specificity of INS for the substrate alanine.…”

“…The bacterial ProRS INS domain has been previously investigated using biochemical, ,, structural, and computational studies . Although there is no available crystal structure of an INS domain-tRNA complex, the computational model of E.…”

“…Interestingly, within the putative INS active site pocket, there are very few amino acid side chains that would be predicted to participate in catalysis. In fact, nearly all of the charged or polar residues in the active site have been investigated by mutagenesis , (Figure ), and the only residues with a significant impact on hydrolytic activity are Lys279, Gly331, and His366. Lys279 is conserved throughout the YbaK superfamily and is believed to bind the A76 phosphate of the tRNA, stabilizing the tRNA conformation. , Gly331 is also conserved throughout the YbaK superfamily and is part of a conserved GXXXP motif, which may play an equivalent role to the oxyanionic hole in serine proteases and d -Tyr-tRNA Tyr deacylases .…”

Role of tRNAPro in pretransfer editing of alanine by prolyl-tRNA synthetase

“…Ïîñòòðàíñôåðíîå ðåäàêòèðîâàíèå èçó÷åíî íàìè ðàíåå ïðè ïîìîùè òåñòà íà äåàöèëèðîâàíèå àëàíèë-òÐÍÊ ÏðîÀëà [17]. Äëÿ ýòîãî ïîäîáðàíà ìóòàíòíàÿ ôîðìà ÏðîÐÑEf Ê279À, õàðàêòåðèçóþùàÿñÿ ïðàê-òè÷åñêèì îòñóòñòâèåì ïîñòòðàíñôåðíîé ðåäàêòèðóþùåé àêòèâíîñòè.…”

“…Ýòîò ðåçóëüòàò ïðÿìî óêàçûâàåò íà ðîëü òÐÍÊ Ïðî â ãèäðîëèçå àëàíèë-àäåíèëàòà. Åãî êèíåòèêà â ïðèñóòñòâèè òÐÍÊ Ïðî ñõîäíà äëÿ äèêîãî òèïà ÏðîÐÑ è ìóòàíòíûõ ôîðì, íåñóùèõ ïîâðåaeäåííûé ðåäàêòèðóþùèé àêòèâíûé öåíòð INS-äîìåíà [17] (ðèñ. 5), ÷òî ñâèäåòåëüñòâóåò î íå-ïðè÷àñòíîñòè ìåõàíèçìà ïîñòòðàíñôåðíîãî ðåäàêòèðîâàíèÿ ê ýòîìó ïðîöåññó.…”