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Biochim Biophys Acta. 2013 Dec;1834(12):2494-501. doi: 10.1016/j.bbapap.2013.08.011. Epub 2013 Sep 6.

A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.

Author information

1
Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China.

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.

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

PMID:
24012560
DOI:
10.1016/j.bbapap.2013.08.011
[Indexed for MEDLINE]
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