THE REQUIREMENT FOR SUBUNIT INTERACTION IN THE PRODUCTION OF PENICILLIUM-CHRYSOGENUM ACYL-COENZYME A-ISOPENICILLIN-N ACYLTRANSFERASE IN ESCHERICHIA-COLI

Subunit interaction in the formation of active acyl-coenzyme A:isopeni cillin N acyltransferase (AT) has been investigated. Various AT deriva tives were produced from altered Penicillium chrysogenum penDE genes p laced in Escherichia coli expression systems. The regions of penDE enc oding the alpha(11 kDa) and beta(29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly(102)/Cys (103). Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing sub units of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of eithe r subunit in the absence of the other did not form active reAT. Howeve r, cotransformation of E. coli with two plasmids, each encoding a diff erent AT subunit, produced reAT having acyl-coenzyme A: 6-aminopenicil lanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing T hr(105) with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subuni t in E. coli produced acyl-CoA:6-APA AT activity. These results sugges t that the formation of reAT involves cooperative folding events betwe en the subunits. In vitro transcription/translation was used to determ ine the origin of the AT hydrolase activity that cleaves the 40-kDa pr ecursor polypeptide. The appearance of a 29-kDa protein (and presumabl y the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT h ydrolysis is an autocatalytic event.