title: Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.
abstract: 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.affiliation: Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. M.D.Lloyd@bath.ac.uk
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Lloyd
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Lipscomb
authors.forename: Kirsty S
authors.initials: KS
authors.lastname: Hewitson
authors.forename: Charles M H
authors.initials: CM
authors.lastname: Hensgens
authors.forename: Jack E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000164:Acremonium / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002511:Cephalosporins / Q000494:pharmacology; D003001:Cloning, Molecular; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; C025071:deacetylcephalosporin C; C025578:deacetoxycephalosporin C; D001216:Asparagine; D014364:Tryptophan; D008715:Methionine; D007501:Iron; D010105:Oxygenases; C045288:deacetoxycephalosporin C hydroxylase; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; C006414:penicillin N; D010100:Oxygen