Controlling the Substrate Selectivity of Deacetoxycephalosporin/deacetylcephalosporin C Synthase

Abstract

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutaratedependent 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 sitedirected 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 (⌬310 mutant) enhanced ring-expansion of penicillin G by ϳ2-fold. A double mutant, ⌬310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The ⌬310/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.

Catalytic Promiscuity in Biocatalysis: Using Old Enzymes to Form New Bonds and Follow New Pathways

“…Single amino acid substitutions can inactivate either activity. [42] In contrast cephalosporin synthesis in prokaryotes uses separate enzymes for the two steps, but both of these enzymes are closely related to the bifunctional one in eukaryotes.…”

Biomimetic Formation of 2‐Tropolones by Dioxygenase‐Catalysed Ring Expansion of Substituted 2,4‐Cyclohexadienones

“…To our knowledge, the only other enzymatic ring expansion that has been demonstrated biochemically is that involved in cephalosporin biosynthesis, which is also catalysed by a nonhaem-iron-dependent oxygenase, deacetoxycephalosporin C synthetase. [19] This unusual transformation thus provides a rationalisation for the biosynthesis of tropolone natural products, and offers another example of the novel chemistry that can be catalysed by non-haem-iron-dependent enzymes in biology.…”

Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production

“…However, it is still not clear how two highly conserved enzymes are able to drive two types of reaction using the same catalytic platform. Although a mutagenic study on fungal DAOC/DACS has identiWed two amino acid positions that are crucial for hydroxylation [58], a similar eVect has not been demonstrated in prokaryotic DACS. Further investigation of DACS hydroxylation activity should provide more insights to the structure-function relationships of DAOCS and DACS.…”

“…The cylinders, arrows, and black lines represent -helices, -sheets, and coil structures, respectively expression in E. coli [28,34,49], S. lividans [22,33], Penicillium chrysogenum [13,24,68,[70][71][72][73], Pseudomonas putida [47], and Pichia pastoris [3]. High level expression of recombinant scDAOCS has often been carried out in E. coli cells together with the use of traditional extraction to purify scDAOCS from E. coli cell lysate for biochemical studies [57,58,90]. However, this often requires multi-step puriWcation procedures that are time-consuming.…”