Complete nucleotide sequence of the penicillin G acylase gene and the flanking regions, and its expression in Escherichia coli.
The pga gene coding for penicillin G acylase (PGA) in Escherichia coli ATCC11105 was cloned, and its complete nucleotide sequence including 5'- and 3'-flanking regions was determined. Two nonidentical subunits that constitute an active PGA enzyme complex are known to be formed by processing of a common precursor molecule [Böck et al., FEMS Microbiol. Lett. 20 (1983) 141-144]. This novel type of protein processing was confirmed by a nucleotide sequencing study together with amino acid sequencing of two PGA subunits. In addition, it was found that the initiation codon, AUG, is preceded by an authentic ribosome-binding site, a consensus promoter sequence and putative cAMP receptor protein (CRP)-binding sites, and that the termination codon, UAA, is followed by a putative transcriptional terminator. The promoter function was confirmed by galactokinase assay using galK fusion plasmids. A recombinant plasmid was constructed to overproduce the enzyme using phage lambda pL promoter. Unexpectedly, thermal induction led to accumulation of the 94-kDa polypeptide rather than active PGA in large amounts. Western immunoblot analysis showed that this large polypeptide is the real precursor of PGA. It is evident, therefore, that the synthesis of active PGA in E. coli is affected by growth temperature and that the precursor processing step(s) is temperature-sensitive.
Oh
S J
SJ
Department of Microbiology, Seoul National University, Korea.
Kim
Y C
YC
Park
Y W
YW
Min
S Y
SY
Kim
I S
IS
Kang
H S
HS
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010399:Penicillin Amidase / Q000235:genetics*; D011487:Protein Conformation
D004792:Enzyme Precursors; D000581:Amidohydrolases; D010399:Penicillin Amidase