从酯酶机制转变为羟基腈裂解酶机制仅需要两个氨基酸替换。
Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions.
作者信息
Padhi Santosh Kumar, Fujii Ryota, Legatt Graig A, Fossum Sara L, Berchtold Reto, Kazlauskas Romas J
机构信息
Department of Biochemistry, Molecular Biology, and Biophysics, and the Biotechnology Institute, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN 55108, USA.
出版信息
Chem Biol. 2010 Aug 27;17(8):863-71. doi: 10.1016/j.chembiol.2010.06.013.
The alpha/beta hydrolase superfamily contains mainly esterases, which catalyze hydrolysis, but also includes hydroxynitrile lyases, which catalyze addition of cyanide to aldehydes, a carbon-carbon bond formation. Here, we convert a plant esterase, SABP2, into a hydroxynitrile lyase using just two amino acid substitutions. Variant SABP2-G12T-M239K lost the ability to catalyze ester hydrolysis (<0.9 mU/mg) and gained the ability to catalyze the release of cyanide from mandelonitrile (20 mU/mg, k(cat)/K(M) = 70 min(-1)M(-1)). This variant also catalyzed the reverse reaction, formation of mandelonitrile with low enantioselectivity: 20% ee (S), E = 1.5. The specificity constant for the lysis of mandelontrile is 13,000-fold faster than the uncatalyzed reaction and only 1300-fold less efficient (k(cat/)K(M)) than hydroxynitrile lyase from rubber tree.
α/β水解酶超家族主要包含催化水解作用的酯酶，但也包括催化氰化物加成到醛上形成碳-碳键的羟基腈裂解酶。在此，我们仅通过两个氨基酸替换就将一种植物酯酶SABP2转化为了羟基腈裂解酶。变体SABP2-G12T-M239K失去了催化酯水解的能力（<0.9 mU/mg），并获得了催化从扁桃腈释放氰化物的能力（20 mU/mg，k(cat)/K(M)=70 min(-1)M(-1)）。该变体还催化了逆反应，以低对映选择性形成扁桃腈：20% ee（S），E = 1.5。扁桃腈裂解的特异性常数比未催化反应快13000倍，且与橡胶树的羟基腈裂解酶相比，催化效率（k(cat/)K(M)）仅低1300倍。
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