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首页> 外文期刊>Biochemistry >Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: The role of alanine 230 in acceptor subsite+1
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Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: The role of alanine 230 in acceptor subsite+1

机译：通过定向进化将环糊精糖基转移酶转化为淀粉水解酶：丙氨酸230在受体亚位点+1中的作用

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摘要

Cycloclextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than trans glycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction. [References: 45]
机译：Cycloclextrin糖基转移酶（CGTase）优选催化转糖基化反应，而许多其他α-淀粉酶家族酶是水解酶。尽管可获得该家族的几种转糖基化酶和水解酶的三维结构，但决定水解和转糖基化特异性的因素尚不清楚。为了鉴定对转糖基化反应特异性至关重要的氨基酸残基，我们进行了容易出错的PCR诱变，并筛选了具有增强水解活性的圆形芽孢杆菌菌株251 CGTase突变体。经过三轮诱变后，水解活性增加了90倍，达到了CGTase报道的最高水解活性。具有最大作用的单个突变（A230V）发生在以前未研究的残基中。此A230V突变体的结构表明，较大的缬氨酸侧链会阻碍底物在受体亚位点+1上的结合，尽管不会达到无法催化的程度。该突变体的水解活性比反式糖基化活性高得多，这特别表明糖受体的使用受到阻碍。该观察结果有利于提出的诱导拟合机制，其中糖受体在受体亚位点+1处的结合在转糖基化中激活了酶[Uitdehaag et al.。 （2000）Biochemistry 39，7772-7780]。由于A230V突变在位点+1处引入了空间位阻，因此预期该突变会对糖受体的使用产生负面影响。因此，突变体A230V的特征强烈支持提出的诱导拟合机制的存在，其中糖受体结合在转糖基化反应中激活CGTase。 [参考：45]

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