title: Computational design of variants for cephalosporin C acylase from Pseudomonas strain N176 with improved stability and activity.
abstract: In this report, redesigning cephalosporin C acylase from the Pseudomonas strain N176 revealed that the loss of stability owing to the introduced mutations at the active site can be recovered by repacking the nearby hydrophobic core regions. Starting from a quadruple mutant M31βF/H57βS/V68βA/H70βS, whose decrease in stability is largely owing to the mutation V68βA at the active site, we employed a computational enzyme design strategy that integrated design both at hydrophobic core regions for stability enhancement and at the active site for activity improvement. Single-point mutations L154βF, Y167βF, L180βF and their combinations L154βF/L180βF and L154βF/Y167βF/L180βF were found to display improved stability and activity. The two-point mutant L154βF/L180βF increased the protein melting temperature (Tm) by 11.7 °C and the catalytic efficiency Vmax/Kmby 57 % compared with the values of the starting quadruple mutant. The catalytic efficiency of the resulting sixfold mutant M31βF/H57βS/V68βA/H70βS/L154βF/L180βF is recovered to become comparable to that of the triple mutant M31βF/H57βS/H70βS, but with a higher Tm. Further experiments showed that single-point mutations L154βF, L180βF, and their combination contribute no stability enhancement to the triple mutant M31βF/H57βS/H70βS. These results verify that the lost stability because of mutation V68βA at the active site was recovered by introducing mutations L154βF and L180βF at hydrophobic core regions. Importantly, mutation V68βA in the six-residue mutant provides more space to accommodate the bulky side chain of cephalosporin C, which could help in designing cephalosporin C acylase mutants with higher activities and the practical one-step enzymatic route to prepare 7-aminocephalosporanic acid at industrial-scale levels.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People's Republic of China. yszhu@tsinghua.edu.cn.|MOE Key Lab of Industrial Biocatalysis, Tsinghua University, Beijing, 100084, People's Republic of China. yszhu@tsinghua.edu.cn.
authors.forename: Yushan
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D002511:Cephalosporins / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods*; D011549:Pseudomonas / Q000201:enzymology*; D013696:Temperature
chemical_list: D002511:Cephalosporins; D050505:Mutant Proteins; C025163:cephalosporin C; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase
keywords: Cephalosporin C acylase; Computational enzyme design; Enzyme engineering; Protein design; Thermostability