Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits.
Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative alpha- and beta-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.
Velasco
J
J
Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain.
Gutierrez
S
S
Campoy
S
S
Martin
J F
JF
D000123:Acetyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000164:Acremonium / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002511:Cephalosporins / Q000096:biosynthesis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008970:Molecular Weight; D009154:Mutation; D011817:Rabbits; D012995:Solubility
D002511:Cephalosporins; D005656:Fungal Proteins; C025163:cephalosporin C; D000123:Acetyltransferases; C018707:acetyl CoA-deacetylcephalosporin C acetyltransferase