Stepwise Loop Insertion Strategy for Active Site Remodeling to

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Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions Md Anarul Hoque, Yong Zhang, Liuqing Chen, Guang-Yu Yang, Mst Afroza Khatun, Hai-Feng Chen, Liu Hao, and Yan Feng ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00018 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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ACS Chemical Biology

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions Md Anarul Hoque†, Yong Zhang†, Liuqing Chen, Guangyu Yang, Mst Afroza Khatun, Haifeng Chen, Liu Hao, Yan Feng* †

These authors contributed equally

* Corresponding author: Yan Feng, Email: [email protected] State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.

ABSTRACT: The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois’s potential for changing enzyme function. By inserting 6 residues into active site loop7, the best variant ML7-B6 demonstrated a 16 fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >107 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has a potential for rapid generation of novel enzyme functions by the loop remodeling.

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The redesign of biocatalysts with high catalytic activity against unnatural substrates is of particular interest, because these methods not only contribute to the understanding of the mechanism of divergent evolution but also broaden the practical applications of biocatalysts in biotechnology.1,2 Different members from the same enzyme family or superfamily usually share key active site residues in a common fold, but exhibit different substrate specificities and varying active-site loops.3-5 Substitutions, insertions and deletions occurring in active-site loops are thought to play a key role in divergent enzyme evolution, which can result in high catalytic activities towards poor or inactive substrates.6-9 Previous studies have achieved enzyme functional changes through site-directed mutagenesis in the active-site loop regions, affecting substrate preference and thermostability as well as altering enzyme enantioselectivity.6,10,11 For instance, iterative saturation mutagenesis (ISM) 10, by grouping individual residues aligning the binding pocket of enzyme correctly into randomization sites and performing saturation mutagenesis iteratively, can accumulate beneficial enatioselectivity mutations, which mainly arise from residue substitutions but not residue insertion or deletion in the active site regions. Relative to site mutagenesis, loop remodeling by residue insertion and deletion of the active site loop regions is generally considered a more efficient method to optimize functional properties because the reconstructed loops are more likely to inhabit conformations that enhance a desired property more than the original scaffold.9,12-16 A recent study showed that the insertion of residues into longer loops near the enzyme active site resulted in dramatic changes to backbone configurations.17-20 Conventional loop insertion methods, such as by grafting loop fragments into the corresponding sites of target proteins, frequently result in misfolded variants with low activity.17,20


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To facilitate active site remolding by residue insertion, we developed a step-wise loop insertion strategy (StLois) (Scheme 1) for efficiently elongating active site loops to produce configurations feasibly favoring novel catalytic reactions of non-native substrates. This strategy uses semi-rational loop insertions that alter the loop backbone in a stepwise manner and compound the fitness effect of mutations. As a first step of StLois, the targeting regions on active site loop can be identified on based of the structural comparison and functional analysis on the counterpart enzymes. Secondly, residue insertions mutagenesis is introduced into the targeting region by constructing saturation mutagenesis libraries with the NNK randomization codon degeneracy. Then mutants with given catalytic properties improvement are selected from libraries. Subsequently, the gene of the best hit used as a template for next round of residue insertion mutagenesis and the process can continue as far as necessary. To rapidly screen mutant libraries, the mutational step length (the number of introduced residues in each round of mutation) is a critical consideration in the stepwise reconstruction of the active site loop of enzyme. The larger the mutational step length, the more dramatic the possible effect on activity. The algorithm Pi=1-(1-Fi)T was used to count the number of transformants for double, triple and quadruple residue insertions/deletions/mutations as a function of NNK codon degeneracy21,22 and to achieve 95% mutation coverage, the size of the smart random saturated library must be 3.0×103, 9.8×104 and 3.1×106 transformants, respectively.22 To avoid a massive, time-consuming library screen, we propose that a saturated mutagenesis library of double residue insertions in the loop would be suitable for each round and that the mutational fitness effect could be compounded to identify stable variants with desired functions. Phosphotriesterase-like lactonases (PLLs) catalyze lactone hydrolysis, including homoserine lactones (HSLs), which serve as bacterial quorum-sensing signals.23 PLLs belong to the 3 ACS Paragon Plus Environment


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amidohydrolase

superfamily

and

contain

an

(α/β)8

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TIM-barrel

structural

fold.23-25

Phosphotriesterases (PTEs) belong to the same superfamily as PLLs and hydrolyze a wide range of hazardous organophosphates (OPs) chemicals with rates approaching the diffusion limit (Kcat /Km∼ 108 M-1s-1 with paraoxon).26 Some promiscuous PLLs also exhibit weak PTE activity.17, 18,23,25,27

In recent studies, loop7 from Pseudomonas diminuta PTE (pdPTE) has been

successfully shortened by loop deletion to expand its lactonase activity, indicating that this loop plays a critical role in substrate specificity.9 However, reverse grafting of the missing PLL loop failed to convert the PTE into an active PLL enzyme.17,19 To investigate the evolutionary route from PLL to PTE and to expand substrate scope of PLL, we used a thermostable PLL from Geobacillus kaustophilus (GkaP-PLL) for active site remodeling using StLois. This strategy allows us to screen a series of relatively small smart mutation libraries that combine the restricted site-saturation mutagenesis on defined positions of the active site loop. Our previous work showed that GkaP-PLL exhibits a similar folded structure as pdPTE but possesses different active loop configurations (Supporting Information Figure S1). Notably, loop7 of GkaP-PLL is 11 amino acids shorter than that of pdPTE (Supporting Information Figure S2). The PTE loop7 has been proposed to contain two possible individual insertion sites, annotated L7-A and L7-B, connected by a 4-amino-acid spacer (Figure 1A).9 There are two more residues in pdPTE L7-A and nine more in L7-B (Figure 1A) than there are in GkaP-PLL loop7. In addition, there are significant differences in the amino acid constitution and spatial architecture (Figure 1B). To efficiently elongate GkaP-PLL loop7, we used the StLois strategy to design and construct smart mutant libraries that introduced two residues in a stepwise manner with degenerate NNK codon randomization (Figure 1C). Two residues were introduced into the GkaP-PLL L7-A region by saturation mutagenesis, and positive variants were screened using OP 4 ACS Paragon Plus Environment

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ethyl-paraoxon as the substrate at 37°C. The most active variant encodes a D and R insertion at positions 235 and 236. This variant, named L7-A2 (DR), exhibited a 2.6-fold increase in catalytic efficiency toward ethyl-paraoxon (kcat/Km of 3.4×102 M-1s-1). Then the variant L7-A2 was used as the starting template for insertion into loop L7-B. A variant with M and I inserted at positions 241 and 242, L7-A2B2 (DR-MI), demonstrated approximately 11-fold improvement in ethyl-paraoxon catalytic efficiency (kcat/Km of 1.4×103 M-1s-1). The feasibility of this strategy was validated by the improved OP hydrolytic activity of the obtained variants. To obtain a more efficient OP hydrolase, we used our previously obtained GkaP-PLL variant 26A8 (ML7, F28I/Y99L/T171S/F228L/N269S/V270G/G273D) and 26A8C (ML8, the ML7 with additional W271C), which demonstrated approximately 38 and 230 times higher PTE activity than the WT,28 as a template for insertion mutagenesis on loop7, respectively. Unfortunately, ML8-derived insertion mutants suffered the protein folding and solubility problem. In contrast, ML7-derived ML7-A2 with a DN insertion showed 116-fold increased activity relative to that of the WT. Additionally, the mutant ML7-B2 with a VN insertion exhibited 6.9 fold activity increase in comparison with the template ML7 variant, that is 261-fold higher catalytic efficiency than the WT (Table 1). Because ML7-B2 (VN) exhibited better OP hydrolysis activity than ML7-A2 (DN), it was chosen for further residue pair insertions on loop7 by StLois. The catalytic efficiency of OP hydrolysis has been determined for all of the variants at 37°C (Table 1). Variant ML7-B6, with a 6-residue insertion (VNLGKY), is the most efficient (kcat/Km=7.9×104 M-1s-1), followed by ML7-B8 (VNLGKYSK) and ML7-B4 (VNLG). These mutants exhibited catalytic efficiencies that were 609-, 469- and 387-fold higher, respectively, than that of the wild-type enzyme. Thus, six residues might be the optimal insertion number for the GkaP-PLL L7 region to achieve PTE catalysis that the variant ML7-B6 could further boost a 5 ACS Paragon Plus Environment


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16 folds hydrolysis activity towards ethyl-paraoxon than that of the 26A8. In contrast, the increase in ethyl-paraoxon hydrolysis activity of the variants was accompanied by a 103- to 104fold decrease in lactonase activity with δ–decanolactone. These results indicate that the obtained variants underwent a shift in substrate specificity, producing enzymes specialized for OP rather than lactone hydrolysis. Recently, critical regions involving in the substrate binding in Sulfolobus solfataricus PLL (SsoPox-PLL) were substituted with 16 conserved residues in PTE family, the resulting chimeric enzyme was unstable and showed a much lower PTE activity than that of the SsoPox.20 By further using a staggered extension process, the most proficient variant with triple mutations(C258L/I261F/W263A) isolated in the optimization showed 4.5 ×104 M-1s-1 catalytic activity at 65ºC on ethyl-paraoxon.20 In addition, a PLL from Deinococcus radiodurans (DrPLL) was directly evolved into 2.0×104 M-1s-1 catalytic activity toward OP compounds.11 Our GkaPPLL variant ML7-B6 indeed has a increase PTE activity in 2.4×105M-1s-1kcat/Km at 65ºC (Table 1), which is around 10 fold higher than the engineered SsoPox-PLL and DrPLL variants. These results suggest that StLois is a powerful technique which might be used for rapid generation of novel PTE enzymes against unnatural substrates. The variants described above were also tested for activity against a variety of commercial OP pesticides; the specific activities of wild-type GkaP and the mutants are shown in Figure 2 and listed in Table S1. All of the obtained variants exhibited significantly higher hydrolytic activities for the tested pesticides (from 17- to 1252-fold compared to the WT). All of the variants showed a higher activity for ethyl-substituted OP derivatives, including ethyl-paraoxon, ethyl parathion, and diazinon, with the exception of the OP chlorpyrifos, which has a polar benzyl group. This preference is distinctly different from that of the WT, which prefers the substrates ethyl-paraoxon, methyl-paraoxon and ethyl-parathion. The elongated loop might 6 ACS Paragon Plus Environment

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provide a favorable active site pocket space and a hydrophobic microenvironment to accommodate non-natural substrates. To address the mechanism of OP hydrolysis with the novel catalysts, we attempted to solve the crystal structures of several variants for structural analysis. However, only variant L7A2B2 was successfully crystallized and determined at a resolution of 1.9 Å (PDB code: 5CH9, Table S2). As shown in Figure 3A, the variant adopts a fold similar to that of the WT, but striking difference a used by the amino acid insertion in loop7 is evident in the active site pocket. The distance between the α metal ion and the loop7 residue V239 in mutant L7-A2B2 (V237 in the WT) is 15.4 Å; the distance is only 10.5 Å in the WT. Additionally, loop3 is moved outward in the L7-A2B2 variant, mainly from residue 98 to105. The width of the main entrance to the active site, as measured from the D73 of loop2 to A105 of loop3, was 9.4 Å in the L7-A2B2 variant but only 2.7 Å in the WT (Figure 3A), indicating an enlargement of the active site entrance caused by the additional loop residues. The pocket volume for substrate binding in L7A2B2 is 1124 Å3 as measured by CASTp online, which is larger than that in WT (430 Å3) and might be more suitable for larger OP substrate catalysis. It has proposed that a closed state of loop7 is optimally pre-organized for paraoxon hydrolysis, but seems to block access to the active site.29 Our previous study has proposed that Y99L mutation in GkaP-PLL result in significant changes in the conformational distribution of loop7 and improvement on its PTE activity.6 Based on the L7-A2B2 structure, the elongated loop7 is found to move upward and may form a dominant open conformation, which could provide the proper steric space for binding diverse OP substrates. To assess the conformational and spatial orientation of the bound substrate, the PTE substrate ethyl paraoxon and lactonase substrate δ-decanolactone were docked into the active 7 ACS Paragon Plus Environment


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sites of GKaP-PLL and mutant L7-A2B2 using Autodock 4.0 (Figure 3C). The substrate ethyl paraoxon binds to the active site in an orientation that polarizes the P-O bond of phosphate esters by ligation of the phosphoryl oxygen with the divalent ions at the β site. The phosphorus atom of ethyl-paraoxon is within 3.7 Å of the β metal in the WT and that is within 3.3 Å in L7-A2B2 mutant. The substrate in the L7-A2B2 mutant is closer to the β-cobalt ion, which would result in strong polarization of the P-O bond and might improve catalysis. In addition, as shown in Figure 3B, the substrate ethyl paraoxon in the L7-A2B2 variant constitutes of a more stable hydrogen bonding network with the residues: Y100, K145, R230 and the D270, compared with that of only K145, R230 in the WT. Three additional stable hydrophobic interactions, involved in H25, F28, and V289, seem to efficiently strengthen its ethyl-paraoxon substrate binding for the L7-A2B2 variant (Supporting Information Figure S3). These results might beneficial for OP pesticides catalysis in the L7-A2B2 variant. The docking of δ-decanolactone resulted in the carbonyl oxygen of the lactone ring directly coordinating with the NH2 atom of R230, which is not present in the mutant L7-A2B2 (Supporting Information Figure S4), suggesting that a relatively weak binding to the lactone happens in the L7-A2B2 mutant. We also performed 80 ns MD simulations on WT and L7-A2B2 mutant complexes. The binding free energy of L7-A2B2 was 28.5±1.3 kcal mol-1, which is 42.3% lower than that of the WT (-20.1±0.8 kcal mol-1). The RMSD and RMSF analyses presented in Supporting Information Figure S5 suggest that the mutant L7-A2B2 is more stable when ethyl paraoxon is present compared to δ-decanolactone. This result suggests that the large pocket in L7-A2B2 is more suitable for a larger OP substrate rather than a smaller lactone substrate. We developed an efficient StLois strategy to address the bottleneck caused by loop elongation in active site remolding to generate novel enzyme functions. Using this methodology, 8 ACS Paragon Plus Environment

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we rapidly compounded the fitness effect of individual mutations by screening smart mutation libraries in a stepwise manner. The synthetic PLL variants exhibited a 600- to 760-fold increase toward a broad scope of OP substrates. The generation of an efficient and broad-spectrum organophosphate hydrolase starting from the thermostable GkaP-PLL scaffold has a potential application in detoxification of environmental OP compounds. Generally, this study demonstrates that StLois is a powerful method for active site remodeling to alter enzyme substrate preference and create new catalysts. The successful change in enzyme function without disrupting the original protein structure has deepened our understanding of the factors that govern molecular evolution in nature. Moreover, this methodology offers an alternative way to design and create novel biocatalysts and even novel functional proteins to perform a wide range of chemical or physiological reactions for which natural enzymes/proteins do not exist.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI. Supporting Information Figures, Tables, and Methods and Materials (PDF)

■ AUTHOR INFORMATION Corresponding Author * Tel.: +86-21-34207189. E-mail: [email protected]

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The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS This work is supported by the grants from Natural Science Foundation of China (31200597， 31620103901, 31570788 ) and Science and Technology Commission of Shanghai Municipality (15JC1400402).

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(20) Del Giudice, I., Coppolecchia, R., Merone, L., Porzio, E., Carusone, T. M., Mandrich, L., Worek, F., and Manco, G. (2015) An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnol. Bioeng. (21) Mena, M. A., and Daugherty, P. S. (2005) Automated design of degenerate codon libraries. Protein Eng. Des. Sel.18, 559-561. (22) Reetz, M. T., Kahakeaw, D., and Lohmer, R. (2008) Addressing the numbers problem in directed evolution. ChemBioChem9, 1797-1804. (23) Afriat, L., Roodveldt, C., Manco, G., and Tawfik, D. S. (2006) The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase. Biochemistry45, 13677-13686. (24) Elias, M., Dupuy, J., Merone, L., Mandrich, L., Porzio, E., Moniot, S., Rochu, D., Lecomte, C., Rossi, M., and Masson, P. (2008) Structural basis for natural lactonase and promiscuous phosphotriesterase activities. J. Mol. Biol.379, 1017-1028. (25) Porzio, E., Merone, L., Mandrich, L., Rossi, M., and Manco, G. (2007) A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus. Biochimie89, 625-636. (26) Omburo, G. A., Kuo, J. M., Mullins, L. S., and Raushel, F. (1992) Characterization of the zinc binding site of bacterial phosphotriesterase. J. Biol. Chem.267, 13278-13283. (27) Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily. J. Biol. Chem.285, 40911-40920. 13 ACS Paragon Plus Environment


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Scheme 1. Stepwise loop insertion strategy (StLois) for active site loops remodeling. In each round, the residue pair is inserted as a function of NNK codon degeneracy. E-A and E-B represent as enzyme A and B, respectively.



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Figure 1.The active-site loop7 of GKaP-PLL and pdPTE, and a model of the engineering stepwise loop insertion approach. A) Refined sequence alignment of the loop7 region of GkaPPLL and pdPTE. The two insertion fragments of loop7 are indicated by L7-A and L7-B. Charged and polar residues are colored in red. B) Overlay of loop7 regions between pdPTE (yellow) and GkaP-PLL (purple). C) The positive mutants with inserted residues obtained by the stepwise loop insertion strategy (StLois).


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Figure 2. Comparison of specific activity for wild-type GkaP-PLL ( (

), ML7-B2 (

), ML7-B4 (

), ML7-B6 (

), and ML7-B8 (

pesticides. The specific activity was measured at 37 °C.


) and the variants ML7 ) against several OP


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 19

Figure 3. Crystal structure analyses of GKaP-PLL and L7-A2B2. A) Overlay of GKaP-PLL (green; PDB code 3TN3) and L7-A2B2 (purple; PDB code 5CH9) structures. The width of the main entrance was measured between D73 of loop2 and A105 of loop3. The major differences between loop7 and loop3 are marked. The metal ions are indicated as α and β. The distances between the α ion and the top part of loop7 are measured. The inserted residues are colored pink. B) Docking poses of GKaP-PLL and mutant L7-A2B2with ethyl paraoxon, which were obtained using autodock 4.0. Ethyl-paraoxon is displayed as balls and sticks and is colored by atom type (gray carbons in WT and yellow carbons in L7-A2B2). The bonding interactions of the substrate with GKaP-PLL or L7-A2B2 are shown as dotted lines. The distances from the ethyl paraoxon phosphorus atom to the β ion is shown as solid lines. C) Active site and substrate binding pocket of GKaP-PLL and the mutant L7-A2B2 in surface view. 18 ACS Paragon Plus Environment

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Table 1.Kinetic parameters of wild-type GkaP and variants[a]

Enzyme

[a]

Phosphotriesterase (ethyl-paraoxon)

Lactonase (δ-decanolactone)

kcat (s-1)

Km (mM)

kcat/Km (M-1s-1)

kcat (s-1)

Km (mM)

kcat/Km (M-1s-1)

(kcat/ Km)PTE/ (kcat/Km) lactonase

WT

0.19±0.01

1.46±0.12

1.3×102 (1)

64.40±1.0

0.08±0.01

7.7×105

1.8×10-4

WT65[b]

0.26±0.13

1.56±0.11

1.6×102

-

-

-

-

L7-A2

0.48±0.07

1.41±0.18

3.4×102 (2.6)

5.74±0.6

0.69±0.11

8.3×103

4.1×10-2

L7-A2B2

1.72±0.05

1.25±0.19

1.4×103 (11)

5.48±0.02

0.82±0.05

6.6×103

2.1×10-1

ML7

3.48±0.11

0.71±0.08

4.9×103 (38)

0.37±0.02

0.22±0.02

1.7×103

5.7×100

ML7-A2

11.14±0.07

0.74±0.10

1.5×104(116)

0.21±0.01

0.70±0.07

2.1×102

7.5×101

ML7-B2

22.46±0.14

0.66±0.12

3.4×104(261)

0.09 ±0.03 0.65 ±0.04 1.1×102

3.3×102

ML7-B4

26.80±0.2

0.52±0.07

5.0×104 (387)

0.02±0.1

0.47±0.05

4.0×101

1.3×103

ML7-B6

30.48±0.51

0.38±0.07

7.9×104 (609)

0.10±0.04

1.87±0.25

5.0×101

1.6×103

ML7-B665[b]

87.33±0.41

0.36±0.11

2.4×105

-

-

-

-

ML7-B8

46.28±0.31

0.76±0.10

6.1×104 (469)

0.09±0.03

1.67±0.21

5.0×101

1.2×103

ML7-B9

16.98 ±1.1

0.60±0.15

2.8×104 (218)

0.11±0.01

2.11±0.32

5.1×101

5.6×102

The Co2+-assembled enzymes were used in kinetic parameter determination. All kinetic data were obtained at 37°C

using average initial velocities that were measured in triplicate, except indicated otherwise. [b]

Kinetic data were examined at 65 ºC.


Stepwise Loop Insertion Strategy for Active Site Remodeling to

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