Characterization of SLAC:: A small laccase from Streptomyces coelicolor with unprecedented activity
Characterization of SLAC:: A small laccase from Streptomyces coelicolor with unprecedented activity
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DOI：
10.1110/ps.04759104
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发表时间：
2004-09-01
期刊：
影响因子：
8
通讯作者：
Canters, GW
中科院分区：
文献类型：
--
作者：
Machczynski, MC;Vijgenboom, E;Canters, GW
漆酶和其他四铜氧化酶通常由三个结构域构成：结构域1和3容纳铜位点，第二个结构域通常有助于形成底物结合裂缝。与此相反，天蓝色链霉菌的基因组被发现编码一个小的，四个铜氧化酶，缺乏第二个结构域。这种蛋白质是一个新的酶家族的代表-双结构域漆酶。相应基因的破坏消除了漆酶在生长培养基中的活性。我们在大肠杆菌中重组表达了这种酶，称为SLAC，并对其进行了表征。该酶结合四个铜离子/单体，紫外可见吸收和EPR测量证实保守的1型铜位点和三核簇是完整的。我们还报告了第一个已知的顺磁NMR光谱的三核铜簇的蛋白质从漆酶家族。该酶是高度稳定的，在煮沸和SDS处理后在变性凝胶中保持二聚体的活性。酶对2，6-二甲氧基苯酚（NH 4）的活性在前所未有的高pH值（9.4）达到峰值，而对亚铁氰化物的活性随pH值降低。SLAC比带正电荷或不带电荷的分子更紧密地结合带负电荷的底物。
Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes-the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.