An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase
An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase
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DOI：
10.1007/s11103-008-9404-7
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发表时间：
2009-01-01
影响因子：
5.1
通讯作者：
Ritsema, Tita
中科院分区：
文献类型：
--
作者：
Altenbach, Denise;Rudino-Pinera, Enrique;Ritsema, Tita
糖苷水解酶家族32（GH 32）具有在其一级结构中高度同源的水解和转糖基化酶。八个氨基酸分散沿着与水解酶或糖基转移酶活性的序列。这些在洋葱液泡转化酶（acINV）中根据在羊茅属蔗糖：蔗糖1-果糖基转移酶（saSST）中的残基发生突变，反之亦然。acINV（W 440 Y）转移酶容量翻倍。相反，saSST（C223 N）和saSST（F362 Y）双重水解。SaSST（N425 S）的水解活性是其转移酶活性的3 - 4倍。有趣的是，根据结晶GH 32酶的3D结构对acINV和saSST进行建模表明，突变saSST（N425 S）、acINV（W 440 Y）和先前报道的acINV（W161 Y）在活性位点口袋入口的表面处非常接近。蔗糖结合盒内外的残基决定了GH 32酶的水解酶和转移酶能力。建模表明，残基分散沿着序列识别受体底物结合的果糖基转移酶的三维结构中的位置。
Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.