ACV synthetase: expression of amino acid activating domains of the Penicillium chrysogenum enzyme in Aspergillus nidulans.

Fragments of ACV synthetase of Penicillium chrysogenum carrying partial activities of amino acid activation were expressed under the alcA promoter in an acvA-deletion mutant of Aspergillus nidulans. The 210 kDa domain A-beta-galactosidase fusion protein was partially cleaved to fragments of 200 and 97 kDa. The domain A fragment and the 312 kDa domain BC construct were identified by peptide specific antibodies and shown to catalyze alpha-aminoadipate-, cysteine-, and valine-dependent ATP/[32P]PPi exchange activity. Substrate specificities were investigated using amino acid analogues. Unexpectedly neither alpha-aminoadipate nor valine activation was exclusive, implying possible misactivations and proof reading functions. Both fragments were only expressed in limited amounts and found to be unstable.