Shimizu Katsuhiko, Morse Daniel E
Tottori University, Tottori, Japan.
University of California at Santa Barbara, Santa Barbara, CA, United States.
Methods Enzymol. 2018;605:429-455. doi: 10.1016/bs.mie.2018.02.025. Epub 2018 Apr 11.
Silicatein, a silica-synthesizing, catalytic triad hydrolase, was discovered in the silica spicules comprising the skeletons of certain marine sponges. Sequence similarity is closest to that of the mammalian cathepsin L, a catalytic triad hydrolase and protease. Genetic substitutions of residues in the catalytic triad, the predictive activities of polymeric and small-molecule analogs of the enzyme, and the wide range of structures accepted as substrates all support a reaction mechanism closely analogous to that established for the classical catalytic triad hydrolases. In this mechanism, hydrogen bonding of residues in the catalytic site is required to enhance nucleophilic attack and consequent hydrolysis of silicon alkoxide (and a wide range of other precursors), enabling subsequent polycondensation. Experimental and computational analyses revealed a novel pathway of self-assembly, in which the silicatein subunits first form a fractally patterned intermediate before entropic rearrangement to the hexagonally close-packed, macroscopic filament that serves both as the catalyst of silica synthesis in the sponge, and as a template guiding the deposition and emergent structure of the macroscopic silica filaments that form the sponge skeleton. Silicatein also proves capable of catalyzing the synthesis of organic silicones, metal oxides, metal phosphates, polylactides, and polymeric materials composed of organic metal compounds from their corresponding precursors, suggesting an evolutionary relaxation of structural substrate specificity that may have been necessary to accommodate the organic adducts of silicic acid suggested to comprise the natural precursor of the biogenic silica. Methods for purification, characterization, assay, and multiple uses of the enzyme are described.
硅酸酶是一种能合成二氧化硅的催化三联体水解酶，在构成某些海洋海绵动物骨架的硅质骨针中被发现。其序列相似性与哺乳动物组织蛋白酶L最为接近，组织蛋白酶L是一种催化三联体水解酶和蛋白酶。催化三联体中残基的基因替换、该酶的聚合物和小分子类似物的预测活性，以及被接受为底物的广泛结构，都支持一种与经典催化三联体水解酶所确立的反应机制极为相似的反应机制。在这种机制中，催化位点中残基的氢键作用对于增强亲核攻击以及随后的硅醇盐（和其他多种前体）水解是必需的，从而实现后续的缩聚反应。实验和计算分析揭示了一种自组装的新途径，其中硅酸酶亚基首先形成一种分形图案化的中间体，然后进行熵重排形成六方密堆积的宏观细丝，该细丝既是海绵中二氧化硅合成的催化剂，又是引导形成海绵骨架的宏观二氧化硅细丝沉积和出现结构的模板。硅酸酶还被证明能够催化由相应前体合成有机硅酮、金属氧化物、金属磷酸盐、聚乳酸以及由有机金属化合物组成的聚合物材料，这表明结构底物特异性在进化过程中有所放宽，这可能是为了适应被认为构成生物源二氧化硅天然前体的硅酸有机加合物所必需的。本文还描述了该酶的纯化、表征、测定及多种用途的方法。
