Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.
Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa.
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AlgL（一种来自铜绿假单胞菌的藻酸盐裂解酶）的功能表征。
DOI：
10.1021/bi301425r
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发表时间：
2012-12-21
期刊：
影响因子：
2.9
通讯作者：
Tipton PA
中科院分区：
文献类型：
--
作者：
Farrell EK;Tipton PA
海藻酸裂解酶（AlgL）通过β-消除反应催化多糖海藻酸的裂解。在铜绿假单胞菌中，algL是藻酸盐生物合成操纵子的一部分，尽管它是藻酸盐生物合成所必需的，但原因尚不清楚。进行稳态动力学研究，以表征其底物特异性，并显示，AlgL操作优先对非乙酰化的海藻酸盐或其前体甘露糖醛酸。成熟的藻酸盐作为部分乙酰化的多糖分泌，因此该观察结果与AlgL用于降解被困在周质空间中的错误定位的藻酸盐的建议一致。反应的kcat/Km随底物中残基的数量线性增加，从含有16个残基的底物的2.1×105 M− 1 s −1增加到含有280个残基的底物的7.9×106 M− 1 s −1。在相同的基底尺寸范围内，kcat在10 s-1和30 s-1之间变化。kcat/Km随底物长度的变化表明AlgL以进行性方式运作。AlgL显示出令人惊讶的缺乏立体特异性，因为它能够催化邻近藻酸盐中的甘露糖醛酸或古洛糖醛酸残基的裂解。因此，该酶能够从甘露糖醛酸和古洛糖醛酸（它们是C5差向异构体）去除C5质子。通过AlgL对藻酸盐的耗尽消化产生二聚体和三聚体产物，其通过1H NMR光谱和质谱表征。快速混合化学淬灭研究表明，没有滞后的二聚体或三聚体的生产，表明AlgL作为一种胞外多糖裂解酶。
Alginate lyase (AlgL) catalyzes the cleavage of the polysaccharide alginate through a β-elimination reaction. In Pseudomonas aeruginosa algL is part of the alginate biosynthetic operon, and although it is required for alginate biosynthesis, it is not clear why. Steady-state kinetic studies were performed to characterize its substrate specificity, and revealed that AlgL operates preferentially on non-acetylated alginate or its precursor mannuronan. Mature alginate is secreted as a partially acetylated polysaccharide, so this observation is consistent with suggestions that AlgL serves to degrade mislocalized alginate that is trapped in the periplasmic space. The kcat/Km for the reaction increased linearly with the number of residues in the substrate, from 2.1×105 M−1s−1 for substrate containing 16 residues to 7.9×106 M−1s−1 for substrate with 280 residues. Over the same substrate size range, kcat varied between 10 s−1 and 30 s−1. The variation in kcat/Km with substrate length suggests that AlgL operates in a processive manner. AlgL displayed a surprising lack of stereospecificity, in that it was able to catalyze cleavage adjacent to either mannuronate or guluronate residues in alginate. Thus, the enzyme is able to remove the C5 proton from both mannuronate and guluronate, which are C5 epimers. Exhaustive digestion of alginate by AlgL generated dimeric and trimeric products, which were characterized by 1H NMR spectroscopy and mass spectrometry. Rapid-mixing chemical quench studies revealed that there was no lag in dimer or trimer production, indicating that AlgL operates as an exopolysaccharide lyase.
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