Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.

Journal article

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.

Authors: Lloyd, MD; Lipscomb, S; Hewitson, K; Hensgens, C; Baldwin, J

• Publication status: Published
• Journal: Journal of biological chemistry
• Volume: 279
• Issue: 15
• Pages: 15420-15426
• Publication date: 2004-04-01
• DOI: 10.1074/jbc.m313928200
• EISSN: 1083-351X
• ISSN: 0021-9258
• Language: English
• Keywords: Tryptophan; Cloning, Molecular; Methionine; Sequence Homology, Amino Acid; Mutagenesis, Site-Directed; Penicillin-Binding Proteins; Protein Binding; Acremonium; Substrate Specificity; Protein Structure, Tertiary; Cephalosporins; Models, Molecular; Penicillins; Mutation; Oxygen; Models, Chemical; Asparagine; Point Mutation; Molecular Sequence Data; Streptomyces; Amino Acid Sequence; Kinetics; Binding Sites; Intramolecular Transferases; Iron; Oxygenases