Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase.
Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.
Loke
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Department of Microbiology, Faculty of Medicine, National University of Singapore, Japan.
Sim
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D000164:Acremonium / Q000201:enzymology*; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014443:Tyrosine / Q000235:genetics*
D005656:Fungal Proteins; D014443:Tyrosine; D010088:Oxidoreductases; C038370:isopenicillin N synthetase