Catalyzes the attachment of isoleucine to tRNA(Ile). As IleRS can inadvertently accommodate and process structurally similar amino acids such as valine, to avoid such errors it has two additional distinct tRNA(Ile)-dependent editing activities. One activity is designated as 'pretransfer' editing and involves the hydrolysis of activated Val-AMP. The other activity is designated 'posttransfer' editing and involves deacylation of mischarged Val-tRNA(Ile) (By similarity). Confers high-level resistance to the antibiotic mupirocin (pseudomonic acid A), an Ile-analog produced by P.fluorescens NCIMB 10586 itself that competitively inhibits activation by Ile-tRNA synthetase, thus inhibiting protein biosynthesis. ATP + L-isoleucine + tRNA(Ile) = AMP + diphosphate + L-isoleucyl-tRNA(Ile) Monomer. IleRS has two distinct active sites: one for aminoacylation and one for editing. The misactivated valine is translocated from the active site to the editing site, which sterically excludes the correctly activated isoleucine. The single editing site contains two valyl binding pockets, one specific for each substrate (Val-AMP or Val-tRNA(Ile)) (By similarity). P.fluorescens NCIMB 10586 possesses two distinct IleRSs (IleRS-R1 and IleRS-R2), each with a different level of sensitivity to mupirocin. Purified IleRs-R2 shows no sensitivity to mupirocin even at a concentration of 5 mM, 100'000 fold higher than the Ki value of IleRS-R1. Belongs to the class-I aminoacyl-tRNA synthetase family. IleS type 2 subfamily.