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

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Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of  -(L-α-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°C. Soluble expression was restored at a reduced temperature of 25°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.Key words: isopenicillin N synthase, site-directed mutagenesis, glycine, Cephalosporium acremonium.

L'isopénicilline N synthase (IPNS) est essentielle à la conversion catalytique du -(L-α-aminoadipoyl)-L-cysteinyl-D-valine en isopénicilline N dans les voies de biosynthèse de la pénicilline et de la céphalosporine. Deux unités de glycine conservées chez l'IPNS de Cephalosporium acremonium (cIPNS), à savoir la glycine-42 et la glycine-256, ont été identifiés par des alignements de séquences et étudiées plus en détails par mutagénèse dirigée afin d'analyser les effets de la substitution sur la catalyse. Notre étude a démontré que les mutations de glycine à alanine autant qu'à sérine ont diminué l'activité catalytique de la cIPNS et ont affecté son expression dans une hôte hétérologue à 37°C. L'expression soluble fut restaurée à une température plus basse de 25°C; il est ainsi possible que ces unités de glycine joueraient un rôle dans le maintien de la structure de protéines locales et seraient indispensables pour l'expression soluble de la cIPNS.Mots clés : isopénicilline N synthase, mutagénèse dirigée, glycine, Cephalosporium acremonium.[Traduit par la Rédaction]

Keywords: Cephalosporium acremonium; glycine; isopenicillin N synthase; isopénicilline N synthase; mutagénèse dirigée; site-directed mutagenesis

Document Type: Research Article

Publication date: October 1, 2001

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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