Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase
العنوان: | Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase |
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المؤلفون: | Charles M. H. Hensgens, Sarah J. Lipscomb, Jack E. Baldwin, Kirsty S. Hewitson, Matthew D. Lloyd, Christopher J. Schofield |
المصدر: | Journal of Biological Chemistry. 279:15420-15426 |
بيانات النشر: | Elsevier BV, 2004. |
سنة النشر: | 2004 |
مصطلحات موضوعية: | Models, Molecular, Penicillin binding proteins, Stereochemistry, Iron, Molecular Sequence Data, Mutant, Streptomyces clavuligerus, Penicillins, Biochemistry, Substrate Specificity, Hydroxylation, chemistry.chemical_compound, Methionine, Penicillin-Binding Proteins, Point Mutation, Amino Acid Sequence, Cloning, Molecular, Intramolecular Transferases, Molecular Biology, chemistry.chemical_classification, Binding Sites, Sequence Homology, Amino Acid, biology, Deacetoxycephalosporin-C synthase, Tryptophan, Active site, Cell Biology, Cephalosporin C, biology.organism_classification, Streptomyces, Cephalosporins, Protein Structure, Tertiary, Acremonium, Oxygen, Kinetics, Enzyme, Models, Chemical, chemistry, Mutation, Mutagenesis, Site-Directed, Oxygenases, biology.protein, Asparagine, Protein Binding |
الوصف: | Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305. |
تدمد: | 0021-9258 |
URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::734c6c04b106b5680015ac23ef2e716e https://doi.org/10.1074/jbc.m313928200 |
حقوق: | OPEN |
رقم الأكسشن: | edsair.doi.dedup.....734c6c04b106b5680015ac23ef2e716e |
قاعدة البيانات: | OpenAIRE |
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