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:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly102/Cys103. Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits 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 either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.
Tobin
M B
MB
Dyson Perrins Laboratory, Oxford, United Kingdom.
Baldwin
J E
JE
Cole
S C
SC
Miller
J R
JR
Skatrud
P L
PL
Sutherland
J D
JD
D000214:Acyl Coenzyme A / Q000235:genetics* / Q000378:metabolism; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D000214:Acyl Coenzyme A; D004271:DNA, Fungal; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase