Mutational studies on triterpene syntheses:: Engineering lupeol synthase into β-amyrin synthase

被引:132
作者
Kushiro, T
Shibuya, M
Masuda, K
Ebizuka, Y
机构
[1] Univ Tokyo, Grad Sch Pharmaceut Sci, Bunkyo Ku, Tokyo 1130033, Japan
[2] Showa Pharmaceut Univ, Machida, Tokyo 1948543, Japan
关键词
D O I
10.1021/ja0010709
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Site-directed mutagenesis was carried out on two triterpene synthases, beta-amyrin (PNY) and lupeol (OEW) synthases, to identify the amino acid residues responsible fbr their product specificity. In addition to sequence comparison among known oxidosqualene cyclases, our previous chimeric studies suggested that (MWCYCR263)-M-258 sequence of beta-amyrin synthase PNY ((MLCYCR260)-M-255 sequence of lupeol synthase OEW) would participate in product differentiation. To test this hypothesis, Trp259 (MWCYCR of PNY) was mutated to Leu (PNY W259L mutant). Functional expression in yeast and product analysis revealed that this mutant produced lupeol as a major product together with beta-amyrin in 2:1 ratio. Some other minor products including butyrospermol were also produced. On the other hand, Leu256 (MLCYCR of OEW) was mutated to Trp (OEW L256W mutant). This mutant produced exclusively beta-amyrin with only minor amount of lupeol, demonstrating that a single mutation had engineered lupeol synthase into beta-amyrin synthase. Therefore, Trp259 of beta-amyrin synthase was identified to be the residue controlling beta-amyrin formation presumably through stabilization of oleanyl cation, while lack of this effect by Leu residue may terminate the reaction at lupenyl cation stage. In further mutation studies, Tyr residue (MWCYCR in PNY and MLCYCR in OEW) conserved in all of the OSCs producing pentacyclic triterpenes was mutated into His which is found in all of those producing tetracyclic carbon skeletons to investigate the role of this Tyr261 of PNY. PNY Y261H mutant produced dammara-18,21-dien-3 beta-ol (as a 3:5 mixture of E/Z isomer at Delta 18) together with a minor amount of dammara-18(28),21-dien-3 beta-ol, demonstrating that Tyr261 of beta-amyrin synthase plays an important role in producing pentacyclic triterpenes presumably by stabilizing one of the cation intermediates generated after dammarenyl cation.
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页码:6816 / 6824
页数:9
相关论文
共 35 条
[1]  
ABE I, 1994, J BIOL CHEM, V269, P802
[2]   ENZYMATIC CYCLIZATION OF SQUALENE AND OXIDOSQUALENE TO STEROLS AND TRITERPENES [J].
ABE, I ;
ROHMER, M ;
PRESTWICH, GD .
CHEMICAL REVIEWS, 1993, 93 (06) :2189-2206
[3]  
Akihisa T, 1997, CHEM PHARM BULL, V45, P2016, DOI 10.1248/cpb.45.2016
[4]  
BUCKINGHAM J, 1998, DICT NATURAL PRODUCT
[5]   Methodology for the preparation of pure recombinant S-cerevisiae lanosterol synthase using a Baculovirus expression system. Evidence that oxirane cleavage and A-ring formation are concerted in the biosynthesis of lanosterol from 2,3-oxidosqualene [J].
Corey, EJ ;
Cheng, HM ;
Baker, CH ;
Matsuda, SPT ;
Li, D ;
Song, XL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1997, 119 (06) :1277-1288
[6]   Studies on the substrate binding segments and catalytic action of lanosterol synthase. Affinity labeling with carbocations derived from mechanism-based analogs of 2,3-oxidosqualene and site-directed mutagenesis probes [J].
Corey, EJ ;
Cheng, HM ;
Baker, CH ;
Matsuda, SPT ;
Li, D ;
Song, XL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1997, 119 (06) :1289-1296
[7]  
GOLLUB EG, 1977, J BIOL CHEM, V252, P2846
[8]   C-13 NMR-SPECTRA OF OLEAN-18-ENE DERIVATIVES [J].
GONZALEZ, AG ;
FRAGA, BM ;
GONZALEZ, P ;
HERNANDEZ, MG ;
RAVELO, AG .
PHYTOCHEMISTRY, 1981, 20 (08) :1919-1921
[9]   Directed evolution to investigate steric control of enzymatic oxidosqualene cyclization. An isoleucine-to-valine mutation in cycloartenol synthase allows lanosterol and parkeol biosynthesis [J].
Hart, EA ;
Hua, L ;
Darr, LB ;
Wilson, WK ;
Pang, JH ;
Matsuda, SPT .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1999, 121 (42) :9887-9888
[10]  
Herrera JBR, 1998, PHYTOCHEMISTRY, V49, P1905, DOI 10.1016/S0031-9422(98)00366-5