Naslov
Serine ordering loop mutants of atypical seryl-tRNA synthetase from Methanosarcina barkeri

Autori
Dulić, Morana ; Gruić-Sovulj, Ita ; Weygand-Durasevic, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts of the HDBMB 2008, Congress of the Croatian Society of Biochemistry and Molecular Biology with international participation / Strelec, Ivica ; Glavaš-Obrovac, Ljubica (ur.). - Osijek : Croatian Society of Biochemistry and Molecular Biology , 2008. 50-50 (ISBN: 978-953-95551-2-0). / - , 2008

Skup
HDBMB 2008 Congress of the Croatian Society of Biochemistry and Molecular Biology with International Participation

Mjesto i datum
Osijek, Hrvatska, 17.09.2008. - 20.09.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
seryl-tRNA synthetase; substrate ordering loop; induced fit; active site mutants

Sažetak
Seryl-tRNA synthetases (SerRS) aminoacylate cognate tRNASer with serine via ATP-dependent pathway. There are two distinct types of SerRS: bacterial or canonical type found in the majority of organisms and “ methanogenic” type confined to the methanogenic archaea. Recently determined crystal structure of methanogenic Methanosarcina barkeri SerRS (mMbSerRS) revealed that binding of serine is accompanied by a notable conformational change in the enzyme active site bringing the “ serine ordering loop” (residues 394-410) in an ordered conformation that consists of an N-terminal  -helix and a loop in the C-terminal part. These induced fit rearrangements seem to be required for proper positioning of the carboxylate oxygen of serine (via direct contact with Gln400) for nucleophilic attack on the  – phosphate of ATP. We designed and kinetically characterized several mutants in the serine ordering loop in order to determine its role in serylation of tRNASer by mMbSerRS. Mutation of Gln400 to Ala did not have large effect on enzyme's efficiency suggesting that the additional contacts with serine ordering loop residues significantly participate in the positioning of serine. To make larger active site, we introduced double mutation Trp396Ala/Gln400Asn (Trp396 packs against the serine amino group closing the binding pocket for serine) which led to three orders of magnitude increase in KM and ten-fold decrease in kcat. Surprisingly, mutation of His250 that is in indirect contact with serine mediated by Gln400 almost abolished enzyme's activity. However, gel-mobility shift analysis indicates that its importance might be in influencing binding of tRNA. In order to study dependence of enzyme's activity on the conformational flexibility of this region, several mutants were designed. Phe397Pro/Ala399Gly double mutant, thought out to prevent formation of  -helix on the N-terminal side of serine ordering loop, showed the huge importance of the  -helix in productive positioning of serine: both kcat and KM for serine were significantly affected. Our attempt to form an  -helix in the C-terminal part of serine ordering loop in Gly402Ala/Gly405Ala mutant led to ten-fold increase in KM, leaving the kcat almost unaffected. Mutation of Pro395, located N-terminally to the  -helix and thus probably influencing its orientation, showed significant, but not drastic drop in enzyme's efficiency implying that there are other interactions involved in correct positioning of the  -helix in the enzyme active site.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA