Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Abstract

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k(cat)/K(m) ratio and 7-fold increase in relative activity.

Deacetoxycephalosporin C synthase (expandase): Research progress and application potential

“…After the initial attempts, more mutations were made at the R308 site. Among the numerous R308 mutants, the relative specificity of the transformation of various penicillin analogues was studied, and the specific activity of R308L, R308V, R308I and R308T against penicillin G was increased by 3.46–7.62 fold [ 35 ]. R308L and R308I revealed the most meaningful improvement in the conversion of penicillin G at about 520% and 760%, respectively, and showed the widest substrate specificity and improved catalytic activity, capable of converting all penicillin analogues in the tests.…”

Iterative Combinatorial Mutagenesis as an Effective Strategy for Generation of Deacetoxycephalosporin C Synthase with Improved Activity toward Penicillin G

“…The mutant enzymes were expressed and purified by similar methods as described by Wu et al (18). The ring expansion activity was assayed in a mixture containing 1 mM dithiothreitol (DTT), 1.8 mM FeSO 4 , 2.56 mM ␣-ketoglutarate, and 4 mM ascorbate with 5.6 mM penicillin G as the substrate in a final volume of 500 l. The reaction mixture was analyzed by highperformance liquid chromatography (HPLC), and the separation and detection conditions were previously described (18). The kinetic parameters of purified wild-type (WT) and mutated enzymes for penicillin G conversion were determined using procedures similar to those described for the enzyme assays, except the reaction time was 30 min.…”