Recombinant Silicateins As Model Biocatalysts in PNAS PLUS Organosiloxane Chemistry

Recombinant silicateins as model biocatalysts in PNAS PLUS organosiloxane chemistry

S. Yasin Tabatabaei Dakhilia,b, Stephanie A. Caslina,b, Abayomi S. Faponlea,c, Peter Quayleb, Sam P. de Vissera,c, and Lu Shin Wonga,b,1

aManchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom; bSchool of Chemistry, University of Manchester, Manchester M13 9PL, United Kingdom; and cSchool of Chemical Engineering and Analytical Science, University of Manchester, Manchester M13 9PL, United Kingdom

Edited by Galen D. Stucky, University of California, Santa Barbara, CA, and approved May 24, 2017 (received for review August 10, 2016) The family of silicatein enzymes from marine sponges (phylum nosiloxanes. Several attempts have been made to use hydrolytic Porifera) is unique in nature for catalyzing the formation of enzymes such as lipases and proteases for the hydrolysis and inorganic silica structures, which the organisms incorporate into condensation of the Si–O bond (11–13). Although they clearly their skeleton. However, the synthesis of organosiloxanes cata- demonstrate the feasibility of the general concept, the range of lyzed by these enzymes has thus far remained largely unexplored. enzymes tested have so far met with only limited success in To investigate the reactivity of these enzymes in relation to this regard to synthetic yield and substrate scope. important class of compounds, their catalysis of Si–O bond hydro- In contrast, poriferans (marine sponges) that use silica as part lysis and condensation was investigated with a range of model of their inorganic skeleton use a family of enzymes termed the organosilanols and silyl ethers. The enzymes’ kinetic parameters silicateins to catalyze the polymerization of soluble silicates into were obtained by a high-throughput colorimetric assay based on silica (14–16). The primary sequences of these enzymes have the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed been reported and they bear a remarkable homology with pro- k K unambiguous catalysis with cat/ m values on the order of teases of the cathepsin family, with ∼65% sequence similarities – −1 μ −1 2 50 min M . Condensation reactions were also demonstrated and ∼50% sequence identities relative to cathepsin L. Both en- by the generation of silyl ethers from their corresponding silanols zymes share a similar Xaa–His–Asn catalytic triad at their active CHEMISTRY and alcohols. Notably, when presented with a substrate bearing site, although in the silicateins a Ser residue occupies the Xaa both aliphatic and aromatic hydroxy groups the enzyme preferen- position rather than Cys in cathepsin L. Previous reports have tially silylates the latter group, in clear contrast to nonenzymatic shown that silicatein-α (Silα), the prototypical member of this silylations. Furthermore, the silicateins are able to catalyze transetherifications, where the silyl group from one silyl ether may be family, can catalyze the hydrolysis of ethoxysilanes such as tet- transferred to a recipient alcohol. Despite close sequence homol- raethoxysilane (TEOS) and triethoxyphenylsilane (17). Because the silicateins have evolved specifically to manipulate ogy to the protease cathepsin L, the silicateins seem to exhibit no – significant protease or esterase activity when tested against anal- the Si O bond, these enzymes may offer a better starting point ogous substrates. Overall, these results suggest the silicateins are for further elaboration into practical biocatalysts in organo- BIOCHEMISTRY promising candidates for future elaboration into efficient and se- siloxane chemistry. This paper outlines the performance of het- α lective biocatalysts for organosiloxane chemistry. erologously produced Sil for both the hydrolysis and condensation of a range of model organosiloxanes. In the process, the devel- silicatein | biocatalysis | organosilicon | organosiloxane | silyl ether opment of a colorimetric high-throughput screening method for silyl ether bond hydrolysis based on the 4-nitrophenoxylate he organosiloxanes, compounds containing C–Si–O moieties, Trepresent a class of compounds with a truly diverse range of Significance applications. They are commonly used in the form of poly- siloxane “silicone” polymers as components of industrial and Organosiloxanes are components in a huge variety of con- consumer products for a variety of purposes such as bulking sumer products and play a major role in the synthesis of fine agents, separation media, protective coatings, lubricants, emul- chemicals. However, their synthetic manipulation primarily sifiers, and adhesives (1–3). Their use as auxiliaries in the relies on the use of chlorosilanes, which are energy-intensive to chemical synthesis of complex molecules is also long-established produce and environmentally undesirable. Synthetic routes (4–6). However, the production and synthetic manipulation of that operate under ambient conditions and circumvent the these compounds are almost entirely dependent on chlorosilane need for chlorinated feedstocks would therefore offer a more feedstocks, which are environmentally undesirable and energy- sustainable route for producing this class of compounds. Here, intensive to produce (7, 8). Furthermore, organosiloxanes, which a systematic survey is reported for the silicatein enzyme, which are entirely anthropogenic in origin, are now known to be per- is able to catalyze the hydrolysis, condensation, and exchange sistent environmental contaminants because little attempt is of the silicon–oxygen bond in a variety of organosiloxanes made to recover and recycle them (3). Synthetic routes that use, under environmentally benign conditions. These results sug- and ultimately recycle, siloxanes and silanols as alternatives gest that silicatein is a promising candidate for development of would in principle be more environmentally sound. selective and efficient biocatalysts for organosiloxane chemistry. One possible strategy toward improved sustainability is to harness enzymes for chemical processing. Such biocatalysts are Author contributions: S.Y.T.D., S.P.d.V., and L.S.W. designed research; S.Y.T.D., S.A.C., and A.S.F. performed research; S.Y.T.D., S.A.C., A.S.F., P.Q., S.P.d.V., and L.S.W. analyzed data; attractive because they offer highly efficient synthesis in terms of and S.Y.T.D., S.A.C., P.Q., S.P.d.V., and L.S.W. wrote the paper. yields and regio- and stereospecificity, together with an ability to The authors declare no conflict of interest. promote reactions under mild conditions and a minimal reliance This article is a PNAS Direct Submission. on halogenated or metallic feedstocks (9, 10). The use of en- Freely available online through the PNAS open access option. – zymes to manipulate the Si O bond would therefore potentially 1To whom correspondence should be addressed. Email: [email protected]. offer more sustainable routes to the synthesis of many com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pounds, as well as for the eventual recycling and reuse of orga- 1073/pnas.1613320114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613320114 PNAS Early Edition | 1of7 Downloaded by guest on September 29, 2021 chromophore is also reported. Additionally, the protease and es- 16000 α terase activity of Sil against analogous substrates is described. 12000 ) Results and Discussion -1 8000

Production and Characterization of Silα. dmol

To acquire this enzyme, a 2 4000 TF-Silα synthetic vector containing cDNA encoding for the mature wild- 0

type Silα from Suberites domencula, fused to an N-terminal (° cm TF-Silα (Mutant-Ser26Ala) hexahistidine tag and codon optimized for expression in Escherichia ME -4000 coli, was used. It is known that the mature form of the protein is -8000 highly hydrophobic and difficult to produce in soluble form (16, 18). -12000 Thus, in attempts to improve its solubility the gene was also subcl- 190 210 230 250 oned with the sequences for a number of proteins known to enhance Wavelength (nm) solubility and folding. Sequences encoding for GST, thioredoxin, small ubiquitin-like modifier, maltose binding protein, or trigger Fig. 2. CD spectra plots of molar ellipticity against wavelength for TF-Silα factor (TF) were inserted between the hexahistidine tag and Silα and TF-Silα(Ser26Ala). and the genes transformed into a variety of E. coli BL21(DE3) strains including Arctic-Express and Origami. Expression trials In contrast, Silα could be maintained in a homogeneous state were then carried out by varying the induction conditions, includ- only at dilute concentrations (micromolar range), which were ing the concentration of the induction agent (isopropyl β-D-1- insufficient for CD measurements. However, analysis by non- thiogalactopyranoside), incubation temperature, and incubation time. denaturing gel electrophoresis for Silα clearly showed a single Overall, these optimization experiments showed that all of the band, indicating nonaggregation (SI Appendix, Fig. S1B). The candidate proteins expressed well in E. coli but only the TF-Silα α protein advanced through the gel at a similar rate as the 25 kDa fusion gave the protein in soluble form. As expected, the Sil reference protein, suggesting that it was of approximately similar (without any fusion tag) was almost entirely insoluble. However, size, globular, and monomeric. it was found that addition of the nondenaturing detergents Tri- ton X-100 and CHAPS into the lysis buffer enabled the recovery Determination of Enzymatic Activity. To confirm that the two of sufficient levels of the soluble protein for some further studies. candidate proteins (Silα and TF-Silα) were catalytically compe- Both the TF-Silα fusion and the solubilized wild-type Silα were tent, their hydrolytic activity against TEOS was tested and the then purified to homogeneity (Fig. 1). amount of precipitated silica quantified by the previously The isolated TF-Silα, and a Ser→Ala mutant at position 26 reported silicomolybdic acid assay (17). The amount of silica (Ser26Ala) of this protein produced using the same procedures, produced upon exposure of silicic acid (derived from acid hy- were fully soluble and could be handled without any special drolysis of TEOS) to these enzymes was also measured. Both precautions. Size-exclusion chromatography of both these pro- assays demonstrated that the enzymes were active for both the teins in isolated form showed a single well-defined species (SI hydrolysis and subsequent condensation of silicate Si–O bonds Appendix, Fig. S1A). The CD spectrum of each protein showed (SI Appendix, Fig. S3). In comparison, the heat-denatured en- clear secondary structural features, indicating the proteins were zyme and chymotrypsin (as a representative serine protease) not denatured or disordered (Fig. 2 and SI Appendix, Fig. S2). displayed only a small amount of nonspecific activity, likely due The spectra of the mutant and unmodified protein essentially to general hydrophobic interactions and nonspecific basic ca- overlap, showing that the mutation did not affect its overall talysis (19). These results were in agreement with previous re- folding. The relative proportions of secondary structures were ports using Silα derived from the native organisms (15, 17) and also calculated from these spectra and were found to be within tests with trypsin and papain that are known to reject TEOS as a 4% of the values calculated from combining the crystallograph- substrate (11, 17). ically derived data of a cathepsin-silicatein chimera (18) and TF (SI Appendix, Tables S1–S3). High-Throughput Colorimetric Assays for Silyl Ether Hydrolysis. To investigate the scope of these recombinant silicateins in the manipulation of a wider range of siloxanes, it was necessary to develop a new screening methodology for this class of com- A 23.1 kDa B 49.6 kDa 23.1 kDa pounds, because the silicomolybdic acid assay is incompatible N- -C N- -C with organosilanes (compounds with C–Si bonds). For this pur- Silα Trigger Factor Silα pose, a series of 4-nitrophenoxy silyl ethers 1–3 was synthesized His His6 6 for use as model substrates (Scheme 1). Here, it was envisaged that hydrolysis of the Si–O bond would result in the release Amount of protein (mg) Amount of protein (mg) of the corresponding silanols 4–6 and the strongly absorbing nitrophenoxylate ion, which can be quantified by UV-visible (UV-Vis) spectrophotometry. 0.3 0.2 0.1 0.05 0.02 0.01 0.002 0.02 0.01 0.005 0.05 0.03 These substrates were used to perform time-course experi- 70 kDa 70 kDa ments with the two enzyme candidates (Fig. 3 and SI Appendix, Figs. S4 and S5) and the data were further verified by GC-MS 25 kDa analysis. It was further observed that the assay in fact generated a yellow color that is visually observable (SI Appendix, Fig. S6), thus making it fully colorimetric rather than only spectrometric. 25 kDa These assays showed the 4-nitrophenoxy substrates are some- what susceptible to hydrolysis, and even in the absence of any Fig. 1. Images of SDS/PAGE gels for Silα (A) and TF-Silα (B) after purification, catalyst an appreciable rate of product formation is detectable. over a range of dilutions, demonstrating the homogeneity of the isolated Nevertheless, assays that used the fully competent enzyme always proteins. Also included above each gel image are the schematic represen- gave enhanced rates of hydrolysis compared with the control tations of the protein constructs. experiments or the uncatalyzed reaction.

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al. Downloaded by guest on September 29, 2021 enzyme could be invoked, although how this effect is related to PNAS PLUS the pH is unclear at present.

Kinetic Analysis of TF-Silα and Silα-Catalyzed Silyl Ether Hydrolysis. To further characterize this enzyme-catalyzed hydrolysis, a series of assays were conducted to extract the Michaelis–Menten kcat and Km values (Table 1 and SI Appendix, Figs. S7–S9). Based on Substrate Abbreviationa R1 R2 R3 Product the pH study above, these assays were performed at pH 8.5 as an 1 TBDMS-ONp Me Me tBu 4 acceptable compromise between relatively good enzyme activity, low levels of background (uncatalyzed) hydrolysis, and avoidance 2 TDS-ONp Me Me Thxa 5 of substrate inhibition. 3 TIPS-ONp iPr iPr iPr 6 In general, the binding of both candidate proteins to all of the substrates was relatively weak, with Km values in the micromolar Scheme 1. Hydrolysis of model 4-nitrophenoxy silyl ethers. aNp, 4-nitrophenyl; range. However, it did not follow in the expected trend of de- TBDMS, tert-butyldimethylsilyl; TDS, thexyldimethylsilyl; Thx, thexyl TIPS, creasing Km with increasing substrate steric bulk. The TDS silyl tri(iso-propyl)silyl. ether 2 gave a lower Km than the smaller tert-butyldimethylsilyl (TBDMS) analog across both TF-Silα and Silα. This observation suggests that the enzymes display some selectivity in regard to Taken together, these results demonstrate the feasibility of substrate shape that is not simply a function of overall steric bulk using 4-nitrophenoxy silyl ethers as a high-throughput assay and of the substrate. The k values also followed this general trend, show that both enzymes accelerated silyl ether bond hydrolysis cat with substrate 2 giving the lowest turnover. significantly above that of background uncatalyzed hydrolysis. The overall catalytic efficiency (kcat/Km) did follow a trend of pH Dependence of TF-Silα Hydrolytic Activity. To investigate the falling with increasing bulk of the substrates. These values were comparable with the data from cathepsin L against its standard effect of pH on enzymatic silyl ether hydrolysis the initial rates of 7 hydrolysis of 1 catalyzed by TF-Silα over pH 6.5–10.5 were de- Cbz-Phe-Arg-NHNp dipeptide nitrophenylanilide substrate (25), suggesting the kinetics data obtained for the silicateins

termined. Here, the initial rates of reaction were used as a CHEMISTRY measure of activity and the net enzyme-catalyzed rate was cal- were plausible. culated after accounting for the background nonenzymatic hy- Ser26Ala Mutation at the TF-Silα Active Site. To confirm that the drolysis. It was found that at low substrate concentrations catalysis did indeed involve the serine residue at the active site, ≤ ( 0.05 mM) higher activities were recorded in the basic pH the Ser26Ala mutant of TF-Silα was then tested. Using TEOS as ∼ A range with a maximum at pH 10 (Fig. 4 ), consistent to the a substrate, only a very small amount of silica was formed, “ ” – optimum pH range of alkaline serine proteases (20 22). This comparable to that of the heat-denatured enzyme and chymo- optimum is related to the ionization state of catalytic His resi- trypsin (SI Appendix, Fig. S3A). These data are in agreement due, which must be unprotonated for activity, and the concen- with a previous report where the equivalent mutation in wild- BIOCHEMISTRY tration of hydroxide ions that participate in the hydrolysis of the type Silα from Tethya aurantia resulted in the abolition of specific acyl-enzyme intermediate (22). catalysis (26). When tested against the chromogenic substrate 2 a Substrate inhibition was observed at lower pH values with this similar result was obtained, with the unmodified TF-Silα result- ∼ effect evident at substrate concentrations above 0.06 mM and ing in a clear positive result whereas only low activity was found ∼ B 0.10 mM at pH 6.5 and 7.5, respectively (Fig. 4 ). This phe- in the mutant, comparable to the other control experiments (Fig. nomenon of substrate inhibition, though not frequently dis- 5A). Although greatly reduced in all cases, total loss of catalysis cussed, is known in many enzyme systems through a variety of is never observed due to residual nonspecific catalysis, as mechanisms (23, 24). Of the proposed mechanisms, the classical previously noted. substrate inhibition model where the substrate acts as an allo- steric regulator is unlikely in this case because 1 bears little re- Molecular Dynamics Modeling of Silα Binding with TBDMS-ONp. It semblance to silicic acid, the presumed natural substrate, and was notable that the enzymes were able to process large non- there are no known compounds bearing C–Si bonds of biological polar organosiloxanes, albeit at a low rate. There is currently no origin (3). Models where the increasing amounts of substrate experimentally derived structure for any of the silicateins, but result in the formation of noncatalytic conformations of the previous models (27) have suggested that the active site pocket

TF-Silα TF-Silα TF-Silα A Silα B Silα C Silα TF-Silα (Mutant-Ser26Ala) TF-Silα (Mutant-Ser26Ala) TF-Silα (Mutant-Ser26Ala) No Enzyme No Enzyme No Enzyme 35 20 10 30 M)

M) 8 μ 25 μ 15 6 20 OH - -OH 10 15 4 TDS TIPS TBDMS-OH 10 5

Concentration (μM) 2 Concentraiton ( 5 Concentration ( 0 0 0 050100150 0 200 400 600 800 1000 0 300 600 900 1200 Time (min) Time (min) Time (min)

Fig. 3. Graphs of the extent of hydrolysis of the 4-nitrophenoxy silyl ether substrates 1 (A), 2 (B), and 3 (C) against time, as measured by UV-Vis absorbance at 405 nm corresponding to the 4-nitrophenoxylate anion (calibrated using data from SI Appendix, Fig. S4). Assays are conducted with 0.00067 mol eq of enzyme relative to 0.1 mM substrate in 5% 1,4-dioxane, 50 mM Tris, and 100 mM NaCl, pH 8.5 at 22 °C.

Tabatabaei Dakhili et al. PNAS Early Edition | 3of7 Downloaded by guest on September 29, 2021 A B pH 8.5 substrate does not fit into this cleft cavity and is, therefore, dis- pH 7.5 placed from the protein surface (SI Appendix, Fig. S13). Never- 0.3 0.3 pH 6.5

) 2 theless, in all cases large structural changes to the protein are ) R = 0.999 −1 −1 recorded, indicating that both substrate and protein must un- dergo significant adjustments to match their shape upon binding. 0.2 0.2 mol min mol min μ μ Silyl Ether Condensation Catalyzed by TF-Silα. It is well-established that hydrolytic enzymes such as proteases and esterases can be 0.1 0.1 driven in the reverse direction (i.e., condensation) by alteration Initial Rate (

Initial Rate ( of the reaction equilibrium (30, 31). To explore the applicability of this general concept to the silicateins, the condensation of 0 0 silanols and alcohols to give the corresponding silyl ethers was 67891011 0 100 200 300 400 500 pH TBDMS-ONp concentration (μM) investigated. For this purpose, the enzymes were lyophilized and the reactions performed in octane. In the first instance, Fig. 4. (A) Graph of the rate of hydrolysis of 1 catalyzed by TF-Silα against condensation of 1-octanol (OcOH, 8) with trimethylsilanol pH with an initial 1 concentration of 0.05 mM. (B) Graph of initial reaction 9 α (TMS-OH, ) to give the corresponding silyl ether (TMS-OOc, velocity for the hydrolysis of 1 catalyzed by TF-Sil against a range of initial 15; Scheme 2) was studied. Analysis of the reaction mixtures at substrate concentrations. The plotted data are obtained after subtraction of the rate of background hydrolysis and calibrated using data from SI Ap- various time intervals by GC-MS showed that the desired pendix, Figs. S4 and S5. The best fit Michaelis–Menten curve (dotted line) product was generated (SI Appendix, Figs. S15 and S16) with and associated R2 value are also shown for the pH 8.5 data. reaction conversions of 20% achieved after 72 h. Control experiments using equimolar amounts of heat-denatured enzyme or when the enzyme was omitted gave only small amounts of of the enzyme is relatively wide, which may allow it accommo- product (3.6% and 1.1%, respectively), via nonspecific catalysis date these larger molecules. or the uncatalyzed reaction. To rationalize the experimental observations, molecular dy- To optimize the condensation reaction, a number of param- namics (MD) simulations with CHARMM were carried out us- eters were investigated. It has previously been reported that ing a homology model constructed from the known crystallographic when used in organic solvents, enzymes lyophilized from buffers structure of cathepsin L (28). The silicatein model structure bound at different pH values gave different activities due to the re- to substrate 1 was created and two sets of MD calculations were tention of the protonation state related to that pH before ly- performed for a period of 1 ns, one with constrained substrate ge- ophilization (32). This effect was investigated for the formation ometry and one fully relaxed structure. After completion of the of 15, using TF-Silα lyophilized from buffered ammonium bi- calculations, the final snapshot of the latter model showed that the carbonate at pH 7, 8, and 9 (SI Appendix, Fig. S18). It was found substrate is bound to the enzyme in close proximity to the active site that the highest conversion was observed with the enzyme sample catalytic triad (Fig. 6 and SI Appendix,Figs.S10–S14). Here, the lyophilized from pH 7. The effect of reactant stoichiometry was binding cavity appears as a wide cleft that is able to accommodate then investigated and a 5 mol. eq. excess of silanol 9 was found to large molecules. The model shows that 1 is oriented such that the Si give the best conversion (SI Appendix, Fig. S19). Much lower atom and its substituent alkyl groups point toward the catalytic conversions were observed with larger excesses of silanol, possibly residues His165 and Ser26. The 4-nitrophenyl group points outward due to the apparent substrate inhibition noted above. Using neat to the solvent and fits between the protein backbone and the Arg146 octanol as the solvent resulted in negligible conversions, thought residue, which projects over the substrate (Fig. 6A). The proximity to be due to the denaturation of the enzyme and displacement of of the phenyl ring face of 1 with the guanidinium terminal of the Arg essential structural water molecules by the alcohol (32). residue (∼3 Å) suggests the intriguing possibility of a cation–π in- To maximize the rate of reaction and to test the thermal sta- teraction (29), which may contribute toward the binding of a hy- bility of the system, the condensations were also carried out at drophobic substrate to the relatively polar binding site. 50 °C and 75 °C. These elevated temperatures gave major im- At the end of the simulation the interatomic distance between provements to conversions and were essentially quantitative after the Si atom and the O atom of Ser26 remains large (∼6 Å) and suggests further molecular motions would be required to enable the attack of the catalytic hydroxy group at the Si atom. The MD TF-Silα No Enzyme (75°C) simulation where the geometry of 1 is constrained shows that the A TF-Silα (Mutant-Ser26Ala) B TF-Silα (75°C) TF-Silα (Heat Denatured) No Enzyme (50°C) TF TF-Silα (50°C) No Enzyme No Enzyme (22°C) Table 1. Table of Michaelis–Menten constants determined for 20 TF-Silα (22°C) Silα and TF-Silα against the model substrates 1–3 (the hydrolysis 18 100 of model peptide 7 by cathepsin L is also listed for comparison) 16 14 80 − − − Enzyme* Substrate K , μM k ,min 1 k /K ,min 1·μM 1 12 m cat cat m 60 10 TF-Silα 1 22.4 ± 2.2 988.3 ± 416.4 55.7 ± 21.2 8 40 TF-Silα 2 8.7 ± 4.5 79.1 ± 19.9 12.3 ± 7.5 6

TF-Silα 3 49.8 ± 17.2 92.1 ± 29.4 2.3 ± 1.4 4 (%) Conversion 20 Silα 1 12.9 ± 2.1 611.1 ± 32.8 48.3 ± 6.0 2 TDS-OH Concentration (μM) TDS-OH 0 α ± ± ± 0 Sil 2 7.5 2.6 61.5 5.4 4.6 1.9 0 400 800 1200 0 1530456075 Silα 3 76.4 ± 47.2 166.5 ± 89.7 4.7 ± 4.0 Time (min) Time (h) † Cathepsin L 7 36 1,200 33.3 Fig. 5. (A) Graph of the extent of hydrolysis of 2 over time catalyzed by *Assays for silicateins performed in 5% vol/vol 1,4-dioxane, 50 mM Tris, and TF-Silα, its Ser26Ala mutant, heat-denatured TF-Silα, and the TF protein 100 mM NaCl, pH 8.5. alone. Assays are conducted under the same conditions as noted for Fig. 3. †Assay performed in 5% vol/vol N,N-dimethylformamide and 100 mM phos- (B) Graph of percentage conversion to silyl ether 15 against reaction time at phate buffer, pH 6.0 (data taken from ref. 25). three different temperatures.

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al. Downloaded by guest on September 29, 2021 A B PNAS PLUS

Arg146

Substrate Product R1 R2 n Product ratio (%)a Conversion (%)a His165 18 21 TES TES 1 2.0 ± 0.05 0.75 ± 0.02 Ser 26 18 22 H TES 1 23.5 ± 2.9 8.5 ± 1.0 18 23 TES H 1 74.3 ± 1.1 26.9 ± 0.42 Fig. 6. Images of substrate 1 bound to Silα at the end of the MD simulation 19 24 TES TES 2 0.8 ± 0.6 0.4 ± 0.3 without substrate constraints. (A) Image of the overall protein structure 19 25 H TES 2 18.4 ± 6.8 9.4 ± 3.4 showing the substrate (green) bound at the large active site cavity and the

overhanging Arg146 (orange). (B) Magnification of the area around the 19 26 TES H 2 80.6 ± 23.3 41.2 ± 11.9 bound substrate, with the substrate (green with the silicon atom in yellow), 20 27 TES TES 3 1.1 ± 0.009 0.4 ± 0.01 Ser26 (magenta), His165 (blue), and Arg146 (orange). Other residues within 5 Å H TES 3 15.6 ± 0.03 6.0 ± 0.01 of the substrate are shown in gray. Asn185 is located behind His165 and is not 20 28 visible from this perspective. 20 29 TES H 3 83.1 ± 1.6 32.1 ± 0.6

a 72 h at 75 °C (Fig. 5B). In comparison, control experiments Scheme 3. Regioselective silyl ether synthesis by silanol condensation. After < 24 h using TF-Silα lyophilized with lyoprotectant mixture, 5 mol eq 11 relative where the enzyme was omitted gave 9% conversion. The ability to diol, 75 °C. of the protein to function at such elevated temperatures is notable but conforms with previous reports that nonpolar organic solvents improve the stability of enzymes through hydrophobic responding ethoxysilanes was investigated. Such intermolecular confinement and thus suppression of denaturation (33, 34). transetherifications would be a useful route to the silylation of Another factor that was investigated was the addition of lyo- more valuable substrates using readily available silyl donors CHEMISTRY protectants such as potassium salts and the crown ether while avoiding the use of chlorosilanes. To demonstrate this 18-crown-6 (18C6) before enzyme lyophilization (33, 35, 36). approach, TMS-OEt, DMPS-OEt, and TES-OEt (12–14,Scheme2) When TF-Silα was colyophilized with KCl and 18C6, then used were used as silyl donors for the silylation of octanol 8 with TF-Silα for the condensation of 15, conversions could be further improved catalysis under the optimized conditions previously used for the compared with when these additives were omitted (SI Appendix, condensation reactions (lyoprotectants added, n-octane, 75 °C). Figs. S20 and S21). Using these optimized conditions, the con- Analysis of the reaction products showed that the silyl groups densation with octanol 8 with further silanol examples, dime- could be successfully transferred (SI Appendix, Figs. S26–S31). thylphenylsilanol (DMPS-OH, 10) and triethylsilanol (TES-OH, However, the reactions were slower compared with the analo- BIOCHEMISTRY 11), were then performed to give the corresponding ethers 16 and gous condensation reactions and generally gave poorer conver- 17 (Scheme 2 and SI Appendix, Figs. S22–S25). In all cases >80% sions (Scheme 2). conversions were achieved, demonstrating the use of this enzyme as a silyl ether condensation catalyst. Regioselective Silylation. The differentiation of hydroxy groups remains an important step toward the chemical synthesis of Silyl Transfer by Transetherification. A major limitation of using complex molecules. Such regioselectivity is typically achieved silanols for condensation is their propensity to form disiloxanes. through the selective deprotection of persilylated substrates (6, As an alternative, the transfer of the silyl group from their cor- 37) but necessitates wasteful global protection beforehand. Di- rect, selective silylation would circumvent the deprotection step and would therefore be more efficient. As an initial investiga- tion into the regioselectivity of biocatalytic silylation, model 4-(ω-hydroxyalkyl)phenol substrates 18–20 that each possess a phenolic and aliphatic alcohol (Scheme 3) were subjected to TF-Silα catalyzed silylation with TES-OH (11). For each of these dihydroxy substrates, analysis of the reaction mixtures indicated the formation of all three possible products (21–29, Scheme 3

a b 1 2 3 4 c and SI Appendix, Figs. S32–S34). Notably, although the enzyme Substrate Abbreviation R R R R Product Time (h) Conv.(%) eventually catalyzed the silylation of both hydroxy groups, in all d 9 TMS-OH Me Me Me H 15 72 99.0 ± 9.6 cases there is an initial preference for the phenolic group. For 10 DMPS-OH Me Me Ph H 16 48 83.0 ± 5.7e 11 TES-OH EtEtEtH 17 72 88.6 ± 8.5 12 TMS-OEt Me Me Me Et 15 72 77.9 ± 7.5 13 DMPS-OEt Me Me Ph Et 16 48 50.0 ± 9.4e 14 TES-OEt Et Et Et Et 17 72 34.0 ± 1.5

Substrate R1 R2 Abbreviationa Scheme 2. Silyl ether synthesis by silanol condensation or ethoxysilane - transetherification. aFive mole equivalents of substrates used relative to 30 OO Piv-ONp octanol. bDMPS, dimethylphenylsilyl; TES, triethylsilyl; TMS, trimethylsilyl. cAs 31 NH NH Piv-NHNp determined by GC-MS quantification. Reaction using TF-Silα lyophilized from 2 the buffered lyoprotectant mixture for 72 h at 75 °C. dReaction using TF-Silα e lyophilized from NH3HCO3 buffer only at 75 °C, shown for comparison. Maximum Scheme 4. Enzyme catalyzed hydrolysis of 4-nitrophenyl pivaloate and conversion achieved after 48 h. pivalamide. aNp, 4-nitrophenyl; Piv, pivaloyl.

Tabatabaei Dakhili et al. PNAS Early Edition | 5of7 Downloaded by guest on September 29, 2021 1, TF-Silα (pH 8.5) 7, TF-Silα (pH 8.5) 1, TF-Silα (pH 8.5) A 1, No Enzyme (pH 8.5) B 7, Chymotrypsin (pH 8.5) C 1, Chymotrypsin (pH 8.5) 30, TF-Silα (pH 8.5) 7, Cathepsin L (pH 6.8) 1, Cathepsin L (pH 6.8) 30, No Enzyme (pH 8.5) 7, No Enzyme (pH 8.5) 1, No Enzyme (pH 6.8) 31, TF-Silα (pH 8.5) 7, No Enzyme (pH 6.8) 1, No Enzyme (pH 8.5) 35 31, No Enzyme (pH 8.5) 15 35 30 30

μM) 25 25 10 20 20 15 15 5 10 10 Concentration (μM) Concentration (μM) Concentration ( 5 5 0 0 0 04080120160 0 50 100 150 050100150 Time (min) Time (Min) Time (min)

Fig. 7. Graphs of the concentration of chromophores (4-nitroaniline and 4-nitrophenoxylate ion) generated against time for the hydrolysis of: (A) TBDMS- ONp (1), Piv-ONp (30), or Piv-NHNp (31) catalyzed by TF-Silα;(B) Cbz-Phe-Arg-NHNp (7) catalyzed by TF-Silα, chymotrypsin, or cathepsin L; and (C) 1 catalyzed by TF-Silα, chymotrypsin, or cathepsin L.

example, the silylation of 19 gave a 52% substrate conversion In contrast, chymotrypsin and cathepsin L only catalyzed the after 24 h, of which over 80% was the aryl siloxane 26. The al- hydrolysis of siloxane 1 at low levels, which is attributable to iphatic siloxane 25 comprised most of the remainder of the nonspecific catalysis (Fig. 7C and Table 2). In all cases, no hy- product, with only trace amounts (∼1%) of the disilylated ma- drolysis of the amide 31 was detectable, although chymotrypsin terial 24. In contrast, negative control experiments where the demonstrated a good level of hydrolytic activity against the ester enzyme was omitted gave very low total conversions and silyla- 30 (Table 2), in agreement with the known promiscuous esterase tion of the aliphatic alcohol was preferred (∼0.5% of 25 was activity of this enzyme even against bulky substrates (39). produced after 24 h), as would be expected due to the higher nucleophilicity of the aliphatic alcohol under these conditions. Conclusions This general trend was repeated for the other substrates that In summary, the production of two recombinant silicateins and a were tested. systematic survey into their reactivity against various organosiloxanes has been conducted. In the process, a high-throughput Comparisons with Small-Molecule Catalysts. Using the condensation colorimetric assay for silyl ether hydrolysis was developed. This reaction of octanol 8 and TMS-OH (9)toform15 as a model assay showed that the enzymes are able to catalyze the hydrolysis reaction, the effectiveness of common catalysts such as imidazole, of a range of silyl ethers, including those with very bulky sub- triethylamine, and histidine were compared. At similar catalyst stituents, albeit at a relatively slow rate. The silicateins display loadings, these bases gave negligible conversions (<0.5% after good activity from pH 7.5–10.0, with substrate inhibition ob- 24 h) compared with the enzyme-catalyzed reaction (17%, SI served below this pH range. Supporting MD modeling of the Appendix,Fig.S35). These small-molecule catalysts also gave low enzyme with a model substrate showed that substrate fits into the regioselectivity with the dihydroxy compound 19.Takingimidaz- binding cavity, with the silicon center oriented toward the pre- ole as an example, if a large amount of catalyst was used (3 mol eq sumed catalytic residues. The binding of these large substrates is relative to alcohol) and the reaction was halted at an early time possible as a result of large conformational changes in the pro- point (after 5 h), the aliphatic silyl ether 24 and disilylated 25 were tein and substrate distortion. These results are consistent with SI Appendix clearly the dominant products ( ,Fig.S36). the proposed hydrolytic mechanism that is used by enzymes These results are significant because they show that the en- possessing a catalytic triad motif. zymatic catalysis is not simply due to the presence of basic func- The silicateins were also shown to catalyze the condensation of tional groups, and that the enzymatic reaction results in contrasting organosilanols and alcohols to give the corresponding silyl ethers regioselectivity compared with small-molecule catalysis. when used in organic solvents. Furthermore, they can catalyze α transetherifications, where the silyl group from one silyl ether Esterase and Protease Activity. Since Sil has a high degree of may be transferred to a recipient alcohol. Notably, when pre- homology with the protease cathepsin L and employs a catalytic sented with a substrate bearing both aliphatic and aromatic hy- triad common to many other hydrolytic enzymes, its esterase and 1 droxy groups the enzyme preferentially catalyzes the silylation of protease activity was surveyed. Here, analogs of where the the latter group, in clear contrast to nonenzymatic silylations. dimethylsiloxy moiety was replaced with an ester or amide (30 and 31 respectively, Scheme 4) were tested. Time-course experiments for the hydrolysis of these substrates catalyzed by TF-Silα and Silα Table 2. Table of percentage conversions for the enzyme showed no specific hydrolysis, comparable with control experi- catalyzed hydrolysis of the various substrates ments where the enzyme was omitted (Fig. 7A and Table 2). Both the candidate enzymes therefore seem to have negligible esterase Net % conversion or amidase activity against such analogous substrates. Enzyme 13031 7 When tested against dipeptide 7 both silicateins also displayed negligible activity in comparison with both chymotrypsin and TF-Silα 19.2 ± 1.2 <0.01 ± 0.0 <0.01 ± 0.0 <0.01 ± 0.0 cathepsin L, which readily hydrolyzed this dipeptide (Fig. 7B and Silα 21.4 ± 0.3 1.3 ± 0.8 <0.01 ± 0.0 <0.01 ± 0.0 Table 2). These results are consistent with a previous report where Chymotrypsin 6.9 ± 0.05 17.5 ± 7.1 <0.01 ± 0.0 21.4 ± 0.5 aCys→Ser mutation in the cathepsin L active site largely abolishes Cathepsin L* 2.5 ± 1.3 1.6 ± 1.7 <0.2 ± 0.01 26.7 ± 1.8 protease activity (38). However, it does not exclude the possibility α Net conversions are calculated after subtraction of background hydrolysis. that Sil may have protease activity against very specific sequences All reactions were performed for 4 h with 0.00067 mol eq enzyme with such as in the autolysis of its own propeptide during maturation of 0.2 mM substrate in 5% vol/vol dioxane, 50 mM Tris, and 100 mM NaCl. the enzyme. Indeed, previous work with the silicatein proenzyme *Assay was conducted at pH 6.8, the optimum for this enzyme; all other has shown that self-cleavage is possible (15). assays were performed at pH 8.5.

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al. Downloaded by guest on September 29, 2021 Despite sequence similarities with the cathepsins, the silica- Further work will be needed to develop an in-depth under- PNAS PLUS teins seem to exhibit no significant protease or esterase activity standing of the structure and mechanism of this family of en- when tested against analogous substrates. The silicateins thus zymes. Such structural elucidation of the silicateins remains a represent an example of divergent evolution where an existing significant challenge (18) due to the low protein production yield ancestral enzyme has evolved to catalyze reactions in a new and their hydrophobicity, but it is anticipated that the work “niche” of chemical space. reported here will provide a solid foundation toward this end and The results reported herein therefore suggest that the silica- to the wider goal of using enzymes for applied biocatalysis. teins are promising candidates for future development into ef- Materials and Methods ficient and selective biocatalysts for organosiloxane chemistry. By providing a new chemical context (e.g., the condensation of The proteins were heterologously produced from synthetic genes in E. coli and isolated using standard procedures. The silyl ether substrates were organic silyl ethers in organic solvents), they may be considered “ ” synthesized from the corresponding chlorosilanes, silyl triflates, or silazanes. prototype silyl etherase enzymes that can be subjected to fur- The GC-MS analyses of the reactions were performed by comparison and ther evolutionary optimization (40). Indeed, powerful directed calibration with chemically synthesized samples. Full details for the materials evolution strategies are now available to generate highly specific and methods are given in SI Appendix. The tabulated CD data are given in and robust biocatalysts for applications in the production of fine Dataset S1. chemicals and functional materials (10, 41). It is envisaged that ACKNOWLEDGMENTS. We thank Emily I. Sparkes for technical assistance such biocatalysts could be used in the chemical synthesis of and Prof. Peter G. Taylor (Open University, Milton Keynes, United Kingdom) complex molecules, where they could be used to selectively in- for assistance with the chemical data analysis. This work was supported by troduce or cleave silyl protecting groups (6) or to recycle these Engineering and Physical Sciences Research Council Grants EP/K011685/1 and relatively expensive silyl groups by transetherification (13). In the EP/K031465/1 (to L.S.W.), Biotechnology and Biological Sciences Research Council Doctoral Training Partnership Graduate Studentship BB/J014478/1 area of materials chemistry, they could be applied for the syn- (to S.A.C.), and a graduate studentship from the Tertiary Education Trust thesis of silicone polymers from nonhalogenated feedstocks (42). Fund of Nigeria (to A.S.F.).

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