Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.
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 deacetoxycephalosp...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
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2004
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| _version_ | 1826268912776380416 |
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| author | Lloyd, MD Lipscomb, S Hewitson, K Hensgens, C Baldwin, J Schofield, C |
| author_facet | Lloyd, MD Lipscomb, S Hewitson, K Hensgens, C Baldwin, J Schofield, C |
| author_sort | Lloyd, MD |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T21:16:50Z |
| format | Journal article |
| id | oxford-uuid:4010a188-b8dd-4d3d-ab3b-8683684eeb4a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:16:50Z |
| publishDate | 2004 |
| record_format | dspace |
| spelling | oxford-uuid:4010a188-b8dd-4d3d-ab3b-8683684eeb4a2022-03-26T14:35:47ZControlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:4010a188-b8dd-4d3d-ab3b-8683684eeb4aEnglishSymplectic Elements at Oxford2004Lloyd, MDLipscomb, SHewitson, KHensgens, CBaldwin, JSchofield, CDeacetoxycephalosporin/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. |
| spellingShingle | Lloyd, MD Lipscomb, S Hewitson, K Hensgens, C Baldwin, J Schofield, C Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title | Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title_full | Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title_fullStr | Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title_full_unstemmed | Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title_short | Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase. |
| title_sort | controlling the substrate selectivity of deacetoxycephalosporin deacetylcephalosporin c synthase |
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