Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase

• Published in: Canadian journal of microbiology Vol. 47; no. 10; pp. 961 - 964
• Main Authors: Loke, P, Sim, T.S
• Format: Journal Article
• Language: English
• Published: Ottawa, ON National Research Council of Canada 01.10.2001; Canadian Science Publishing NRC Research Press
• Subjects: Acremonium - enzymology; Acremonium strictum; Alanine - genetics; Amino Acid Sequence; Amino acids; Bacteriology; Biological and medical sciences; Catalysis; Catalysts; Cephalosporium acremonium; DNA Mutational Analysis; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; genes; Genetics; glycine (amino acid); Glycine - genetics; Isopenicillin N synthase; Microbiology; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; Mutation; Oxidoreductases - genetics; Oxidoreductases - metabolism; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Proteins; Sequence Alignment; Serine - genetics; site-directed mutagenesis; Temperature; Enzyme; β-Lactams; Acremonium strictum; Sequence alignment; Cephalosporium; Site directed mutagenesis; Biosynthesis; Synthase; Glycine; Conversion; Fungi; Penicillin derivatives; Valine; Aminoacid; Neurotransmitter; Fungi Imperfecti; Mutation; Thallophyta
• ISSN: 0008-4166; 1480-3275
• DOI: 10.1139/cjm-47-10-961

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.