Recombinant silicateins as model biocatalysts in organosiloxane chemistry

Abstract

The family of silicatein enzymes from marine sponges (phylum Porifera) is unique in nature for catalyzing the formation of inorganic silica structures, which the organisms incorporate into their skeleton. However, the synthesis of organosiloxanes catalyzed by these enzymes has thus far remained largely unexplored. To investigate the reactivity of these enzymes in relation to this important class of compounds, their catalysis of Si-O bond hydrolysis and condensation was investigated with a range of model organosilanols and silyl ethers. The enzymes' kinetic parameters were obtained by a high-throughput colorimetric assay based on the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed unambiguous catalysis with k cat /K m values on the order of 2-50 min. Condensation reactions were also demonstrated by the generation of silyl ethers from their corresponding silanols and alcohols. Notably, when presented with a substrate bearing both aliphatic and aromatic hydroxy groups the enzyme preferentially silylates the latter group, in clear contrast to nonenzymatic silylations. Furthermore, the silicateins are able to catalyze transetherifications, where the silyl group from one silyl ether may be transferred to a recipient alcohol. Despite close sequence homology to the protease cathepsin L, the silicateins seem to exhibit no significant protease or esterase activity when tested against analogous substrates. Overall, these results suggest the silicateins are promising candidates for future elaboration into efficient and selective biocatalysts for organosiloxane chemistry.silicatein | biocatalysis | organosilicon | organosiloxane | silyl ether T he organosiloxanes, compounds containing C-Si-O moieties, represent a class of compounds with a truly diverse range of applications. They are commonly used in the form of polysiloxane "silicone" polymers as components of industrial and consumer products for a variety of purposes such as bulking agents, separation media, protective coatings, lubricants, emulsifiers, and adhesives (1-3). Their use as auxiliaries in the chemical synthesis of complex molecules is also long-established (4-6). However, the production and synthetic manipulation of these compounds are almost entirely dependent on chlorosilane feedstocks, which are environmentally undesirable and energyintensive to produce (7,8). Furthermore, organosiloxanes, which are entirely anthropogenic in origin, are now known to be persistent environmental contaminants because little attempt is made to recover and recycle them (3). Synthetic routes that use, and ultimately recycle, siloxanes and silanols as alternatives would in principle be more environmentally sound.One possible strategy toward improved sustainability is to harness enzymes for chemical processing. Such biocatalysts are attractive because they offer highly efficient synthesis in terms of yields and regio-and stereospecificity, together with an ability to promote reactions under mild conditions and a minimal reliance on halogenated or metallic feedstocks (...

Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins

“…Müller et al . demonstrated that the same silicatein could interact with bulky bis(p-aminophenoxy)-dimethylsilane which contains two Si-O and two S-C bonds, that confirms that silicatein could interact not only with orthosilicic acid itself 10 , 16 . Besides that, silicateins possess directly confirmed proteolytic activity, which is hydrolytic too 17 – 19 .…”

“…Cysteine in the active site is supposed to be non-efficient for silica polymerization catalysis and is commonly described as a feature of cathepsins L 3 . Site-directed mutagenesis confirmed the crucial role of Ser26 and His165 in silicateins 7 , 10 .

Figure 1 Previously proposed mechanisms of the silicatein activity.

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“…Previous works on silicatein mutagenesis demonstrated a key role of Ser26 and His165 from the cathepsin-like catalytic triad, since S26A and H165A mutants failed to polymerize silica 7 , 10 . Both groups used tetraethyl orthosilicate (TEOS) as a silica acid precursor, which is poorly hydrolyzed in water at neutral pH.…”

“…Dakhili and coworkers studied silicatein α of S . domuncula with 4-nitrophenyl pivaloate or pivalamide as a substrate 10 , 16 . These compounds do not undergo further condensation, so, the experiment proved that silicatein is able to catalyze the hydrolysis reactions.…”

Quantum Mechanics/Molecular Mechanics Studies on the Relative Reactivities of Compound I and II in Cytochrome P450 Enzymes

“…Two different approaches were used in this work, namely (1) DFT studies on enzyme active site models and (2) QM/MM calculations on a full enzymatic structure of a P450 isozyme. These QM/MM methods have been described in details elsewhere [ 55 , 56 , 62 , 63 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 ] and were extensively benchmarked and calibrated against experimental data. In particular, rate constants of small model complexes were reproduced excellently as compared to experimental data for oxygen atom transfer by iron (IV)-oxo and iron (IV)-imido species [ 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 ].…”