Kinetic characterization of homologously overexpressed isoleucyl-tRNA synthetates in Bacillus megaterium

Izoleucil–tRNA–sintetaze (IleRS) su enzimi koji u dva koraka kataliziraju nastajanje kovalentne veze između izoleucina (Ile) i pripadne molekule tRNAIle u procesu biosinteze proteina. U prvom koraku, koji se naziva aktivacija, nastaje izoleucil–adenilat dok se u drugom koraku aktivirana aminokiselina prenosi na molekulu tRNAIle. Postoje dva tipa IleRS koji se razlikuju po strukturnim karakteristikama i rezistenciji na antibiotik mupirocin. Genom bakterije Bacillus megaterium neuobičajeno sadrži gene za oba tipa IleRS, što čini navedene proteine zanimljivim za istraživanje. U sklopu ovog rada, proteini IleRS1 i IleRS2 dobiveni su homolognom nadekspresijom u bakteriji B. megaterium te pročišćeni afinitetnom kromatografijom. Molekula tRNAIle heterologno je nadeksprimirana u bakteriji Escherichia coli i pročišćena kromatografijom obrnutih faza. Kinetička karakterizacija navedenih enzima je pokazala da u aktivaciji IleRS2 ima oko 25 × veći KM (Ile) od IleRS1. Međutim, u ukupnoj reakciji aminoacilacije, koja uključuje i molekulu tRNA, dolazi do znatne promjene KM (Ile) za oba enzima što rezultira većim KM (Ile) za IleRS1 u odnosu na IleRS2. To sugerira da interakcija s tRNA različito utječe na mehanistička svojstva IleRS1 i IleRS2.Isoleucyl–tRNA synthetases (IleRS) are enzymes that catalyze covalent attachment of amino acid isoleucine to the cognate tRNAIle during protein biosynthesis. The first step of the reaction (activation) results in the formation of isoleucyl adenylate, while the second step comprises the transfer of activated isoleucine to the cognate tRNAIle. There are two types of IleRSs that differ mainly in structural elements and resistance to antibiotic mupirocin. The genome of Bacillus megaterium uncommonly contains genes encoding both IleRSs making these proteins interesting for studying. Within this work, proteins IleRS1 and IleRS2 were homologously overexpressed in B. megaterium and purified by affinity chromatography. The tRNAIle molecule was overexpressed in Escherichia coli and purified by reverse–phase chromatography. The kinetic characterization of both enzymes showed that, in the activation step, IleRS2 has 25 × higher KM (Ile) than IleRS1. In overall aminoacylation, which includes the tRNA, the KM (Ile) values change for both enzymes, resulting in the higher KM (Ile) for IleRS1 than for IleRS2. The obtained results suggest that interaction with the tRNA promotes a different influence on the mechanistic properties of IleRS1 and IleRS2