title: A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.
abstract: A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.
authors.affiliation: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Jianping
authors.initials: J
authors.lastname: Wu
authors.forename: Lirong
authors.initials: L
authors.lastname: Yang
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D008610:Menthol / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D044202:Pseudomonas alcaligenes / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008610:Menthol; D008049:Lipase
keywords: 5,5′-dithiobis-(2-nitrobenzoic acid); Chemical modification; DTNB; Diastereopreference; Lipase; MALDI-TOF; MD; PAL; Pseudomonas alcaligenes lipase; Site-directed mutagenesis; Steric exclusion; Structural rigidity; WT; matrix assisted laser desorption/ionization time of flight; molecular dynamic; wide type