Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3-bromo-2-methylpropanoate and ethyl 3-phenylbutyrate

被引:72
作者
Horsman, GP
Liu, AMF
Henke, E
Bornscheuer, UT
Kazlauskas, RJ
机构
[1] McGill Univ, Dept Chem, Montreal, PQ H3A 2K6, Canada
[2] Ernst Moritz Arndt Univ Greifswald, Inst Chem & Biotechnol, Dept Tech Chem & Biotechnol, D-17487 Greifswald, Germany
关键词
directed evolution; enantioselectivity; hydrolases; mutagenesis; screening;
D O I
10.1002/chem.200204551
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E = 17). Previous directed evolution identified mutants Asp158Asn and Leu181GIn that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 Angstrom from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.
引用
收藏
页码:1933 / 1939
页数:7
相关论文
共 84 条
[11]  
BURROWS J, 2001, Patent No. 066525
[12]   QUANTITATIVE-ANALYSES OF BIOCHEMICAL KINETIC RESOLUTIONS OF ENANTIOMERS [J].
CHEN, CS ;
FUJIMOTO, Y ;
GIRDAUKAS, G ;
SIH, CJ .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1982, 104 (25) :7294-7299
[13]   Enzyme engineering: rational redesign versus directed evolution [J].
Chen, RD .
TRENDS IN BIOTECHNOLOGY, 2001, 19 (01) :13-14
[14]   Structural constraints in protein engineering - The coenzyme specificity of Escherichia coli isocitrate dehydrogenase [J].
Chen, RD ;
Greer, AF ;
Dean, AM .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1997, 250 (02) :578-582
[15]   CLONING AND NUCLEOTIDE-SEQUENCE OF AN ESTERASE GENE FROM PSEUDOMONAS-FLUORESCENS AND EXPRESSION OF THE GENE IN ESCHERICHIA-COLI [J].
CHOI, KD ;
JEOHN, GH ;
RHEE, JS ;
YOO, OJ .
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, 1990, 54 (08) :2039-2045
[16]   Asymmetric Diels-Alder, Michael, and Aldol reactions using a planar chiral 1,3-oxazol-2(3H)-one derived from (R)-(+)-4-hydroxy-[2.2]paracyclophane [J].
Cipiciani, A ;
Fringuelli, F ;
Piermatti, O ;
Pizzo, F ;
Ruzziconi, R .
JOURNAL OF ORGANIC CHEMISTRY, 2002, 67 (08) :2665-2670
[17]  
Clark D., 2000, [No title captured], Patent No. [00/49005, 0049005]
[18]   SYNTHESIS OF THE S-ENANTIOMER OF PANICULIDINE-A - ABSOLUTE R-CONFIGURATION OF THE NATURAL PANICULIDINE-A AND PANICULIDINE-B [J].
CZESKIS, BA ;
MOISSENKOV, AM .
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1, 1989, (07) :1353-1354
[19]   4-SUBSTITUTED-5,5-DIMETHYL OXAZOLIDIN-2-ONES AS EFFECTIVE CHIRAL AUXILIARIES FOR ENOLATE ALKYLATIONS AND MICHAEL ADDITIONS [J].
DAVIES, SG ;
SANGANEE, HJ .
TETRAHEDRON-ASYMMETRY, 1995, 6 (03) :671-674
[20]   Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces [J].
Dean, JLE ;
Colfen, H ;
Harding, SE ;
Rice, DW ;
Engel, PC .
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, 1997, 25 (5-6) :417-422