Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity |
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Authors: | Wu Xiao-Bin Tian Xiu-Yun Ji Jun-Jie Wu Wei-Bin Fan Ke-Qiang Yang Ke-Qian |
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Institution: | (1) State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People’s Republic of China;(2) National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China; |
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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 kcat/Km ratio and 7-fold increase in relative activity. |
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