Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase.
Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37 degrees C. Soluble expression was restored at a reduced temperature of 25 degrees C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.
Loke
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Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore
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D000164:Acremonium / Q000201:enzymology*; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D005998:Glycine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics; D013696:Temperature
D012694:Serine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000409:Alanine; D005998:Glycine