Role of Trp140 at subsite −6 on the maltohexaose production of maltohexaose‐producing amylase from alkalophilic Bacillus sp.707
Role of Trp140 at subsite −6 on the maltohexaose production of maltohexaose‐producing amylase from alkalophilic Bacillus sp.707
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DOI：
10.1110/ps.051877006
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发表时间：
2006-03
期刊：
影响因子：
8
通讯作者：
R. Kanai;K. Haga;T. Akiba;K. Yamane;K. Harata
中科院分区：
文献类型：
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作者：
R. Kanai;K. Haga;T. Akiba;K. Yamane;K. Harata
来自嗜碱芽孢杆菌属707的产麦芽六糖淀粉酶（G6-淀粉酶）主要产生麦芽六糖（G6），产率为来自短链直链淀粉（DP = 17）的总产物的>30%。我们之前的晶体学研究表明，G6-淀粉酶有9个亚位点，从−6到+3，并指出了Trp 140的吲哚部分在G6生产中的重要性。G6-淀粉酶对短于麦芽七糖的寡糖具有非常低水平的水解活性。为了阐明G6产生的机制，我们确定了G6-淀粉酶与G6和麦芽五糖（G5）复合物的晶体结构。在G6-淀粉酶/G5复合物的活性位点中，G5与亚位点−6至−2结合，而G1和G6分别位于G6-淀粉酶/G6复合物的亚位点+2和−7至−2。在这两种结构中，位于亚位点-6的葡萄糖基残基与Trp 140的吲哚部分堆叠在4 π的距离内。当Trp 140被亮氨酸（W140 L）或酪氨酸（W140 Y）替代时，突变酶的活性的测量显示，通过W140 L由短链直链淀粉产生的G6产量低于通过W140 Y或野生型酶的产量。Trp 140和底物糖之间的面对面的短暂接触被认为是调节葡萄糖基残基在亚位点-6的分布，并控制G6生产的产品特异性。
Maltohexaose‐producing amylase (G6‐amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short‐chain amylose (DP = 17). Our previous crystallographic study showed that G6‐amylase has nine subsites, from −6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6‐amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6‐amylase complexes with G6 and maltopentaose (G5). In the active site of the G6‐amylase/G5 complex, G5 is bound to subsites −6 to −2, while G1 and G6 are found at subsites + 2 and −7 to −2, respectively, in the G6‐amylase/G6 complex. In both structures, the glucosyl residue located at subsite −6 is stacked to the indole moiety of Trp140 within a distance of 4Å. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short‐chain amylose by W140L is lower than that by W140Y or wild‐type enzyme. The face‐to‐face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite −6 and to govern product specificity for G6 production.