Enhanced Î±-Zearalenol Hydrolyzing Activity of a Mycoestrogen

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Enhanced #-zearalenol hydrolyzing activity of a mycoestrogendetoxifying lactonase by structure-based engineering Zhongxia Xu, Weidong Liu, Chun-Chi Chen, Qian Li, Jian-Wen Huang, Tzu-Ping Ko, Guizhi Liu, Wenting Liu, Wei Peng, Ya-Shan Cheng, Yun Chen, Jian Jin, Hua-Zhong Li, Yingying Zheng, and Rey-Ting Guo ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01826 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Enhanced α-zearalenol hydrolyzing activity of a mycoestrogen-detoxifying lactonase by structure-based engineering Zhongxia Xu,1,2* Weidong Liu,1* Chun-Chi Chen,1* Qian Li,1 Jian-Wen Huang,3 Tzu-Ping Ko,4 Guizhi Liu,1 Wenting Liu,1 Wei Peng,1 Ya-Shan Cheng,1 Yun Chen,5 Jian Jin,5 Huazhong Li,2** Yingying Zheng,1** Rey-Ting Guo1** 1

Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial

Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China 2

School of Biotechnology, Jiangnan University, Wuxi 214122, China

3

AsiaPac Biotechnology Co., Ltd, Dongguan 523808, China

4

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

5

School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China

*ZX, WL and CCC contribute equally. **Corresponding authors: [email protected] (RTG); [email protected] (YZ); and [email protected] (HL)

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Abstract The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate but ZOL, especially the α-form, shows higher estrogenic toxicity than ZEN. To enhance α-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against α-ZOL, with about 2.7-fold reduction in substrate affinity but a 5.2-fold higher turn-over rate. We then determined two V153H/ZOL complex structures. Here the α-ZOL lactone ring is hydrogen bonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity towards the more toxic α-ZOL, with great potential in further industrial applications.

Key words: mycoestrogen-detoxifying lactonase; zearalenone; α-zearalenol; complex structure; rational design; substrate specificity


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1 Introduction Zearalenone (ZEN) is a mycoestrogenic resorcylic acid lactone which is produced by Fusarium species and widely detected in musty corn, wheat and other cereals. ZEN can cause reproductive disorders of swine and other domestic animals, giving rise to a huge loss in feed and breeding industry.1, 2 The molecular structure of ZEN contains a resorcylic acid scaffold, which is linked to a 14-membered macrolactone moiety (Fig. 1A). To date, six major natural derivatives of ZEN have been isolated, and the major species is zearalenol (ZOL).3 ZOLs exist as two isoforms, α-ZOL and β-ZOL, and differ from ZEN only by the functional group on C6’ (Figs. 1B and 1C). In vitro breast cancer cell stimulation experiments indicate that the α-ZOL exhibits 92-fold higher estrogenicity comparing to ZEN. On the other hand, β-ZOL is less estrogenic which only exhibits half of the ZEN activity.4 In a rat uterus bioassay, the α-ZOL isomer was three times more estrogenic than ZEN while the β-ZOL showed equal activity as ZEN.5 Moreover, ZOLs not only exist as natural derivatives, but can also be transformed from ZEN by some livestock.6, 7

Since ZEN and its derivatives can bring huge loss to breeding industry and has negative effects on human health, increasing attention has been paid to develop ZEN detoxification strategies.8 Physical absorption and chemical degradation are popular methods to detoxify the toxin-contaminated feedstocks.8, 9 Though certain effects can be achieved, these methods are non-selective and might reduce the nutritional factors in the feeds. In comparison, using toxin-degrading enzymes which achieves specific


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detoxification is a more attractive alternative.10-12 A promising ZEN degrading enzyme denoted ZHD101 has been identified in Clonostachys rosea and widely studied.10, 13-16 ZHD101 cleaves the ester bond of the macrolactone of ZEN and yields non-toxic alkylresorcinol product (Fig. 1A). ZOLs are hydrolyzed by ZHD101 via the same mechanism as ZEN, yielding the non-toxic products. However, the activity of ZHD101 against ZOLs was only ~40% of that against ZEN. A low activity against α-ZOL normally gives rise to an incomplete detoxification of feed contaminated by ZEN and its derivatives. Improving the activity of ZHD101 against α-ZOL is thus essential to promote its application as a safeguard of feed.

Recently, we solved the complex structure of ZHD101 and ZEN.17 ZHD101 adopts a core α/β-hydrolase domain, and the catalytic center is composed of a Ser-His-Glu triad. Through the high-resolution crystal structure, we clearly observed the substrate-binding network in the active site and proposed several residues that are critical for the enzyme activity. Here, we further report the crystal structures of ZHD101 in complex with α-ZOL and β-ZOL to illustrate their binding modes. By analyzing

these

complex

structures,

several

residues

surrounding

the

substrate-binding site were identified and the enzyme was engineered in an attempt to enhance the hydrolyzing activity towards the more toxic α-ZOL. We produced 23 mutants, among which V153H showed the highest increase in α-ZOL hydrolysis while preserving its activity against ZEN. The V153H mutant was then carefully characterized and its crystal structures in complex with ZOLs were also determined to


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elucidate the molecular mechanism.

2 Methods 2.1 Protein expression, purification and mutagenesis The gene encoding the inactive ZHD101 mutant S102A was constructed into pET-46 Ek/LIC vector and expressed in Escherichia coli BL21 (DE3) by using IPTG induction. The recombinant protein with an N-terminal His-tag was purified via Ni-NTA metal-affinity and DEAE ion-exchange chromatography. Following dialysis in a buffer of 150 mM NaCl and 25 mM Tris, pH 7.5, the protein was concentrated to 100 mg/ml for storage.

Site-directed mutagenesis was carried out by using QuickChange Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA), with the plasmid containing the zhd101 gene as the template and the mutagenic primers as listed in Table S1. After verification by sequencing, the correct plasmids were transformed into E. coli for expression. The ZHD101 mutant proteins were expressed and purified with the same procedures as those of the inactive mutant S102A, and their expression levels and purity were similar to those of S102A. Like the wild-type protein, the His-tag did not affect the expression levels of mutants.

2.2 Enzymatic assays The enzyme activity was characterized by substrate depletion. Each assay solution


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(210 µl) contained 5 µl substrate (5 mg/ml ZEN or 5 mg/ml ZOL) and 5 µl enzyme (0.25 mg/ml) in a buffer of 25 mM Tris and 150 mM NaCl, pH 7.5. After incubation for 10 min at 30 ºC, 50 µl 1 N HCl and 300 µl methanol were added to terminate the reaction. After filtration, 20 µl assay solution was analyzed by using a high-performance liquid chromatography system (HPLC, Agilent 1200) equipped with a Welch Ultimate XB-C18 column (4.6 mm × 250 mm, 5 µm, Welch Materials, Inc., Shanghai, China). The sample was eluted by 60% acetonitrile at the rate of 0.6 ml/min and the absorbance was detected at 254 nm. The amounts of residual substrate were calculated according to the peak area under the HPLC elution curve. The enzyme activity is presented as the decreased amount of substrate per minute.17

The Michaelis-Menten constants for α-ZOL substrate were determined by using 0.2 µM enzyme and varying concentrations of the α-ZOL substrate (3.7 µM - 222.9 µM for wild-type assay and 18.6 µM - 594.5 µM for the mutants). The Michaelis-Menten constants for β-ZOL as a substrate were determined by using 0.3 µM enzyme and varying concentrations of β-ZOL (4.4 µM - 140.8 µM). All data were obtained in triplicate and the average values were adopted. Km and kcat were determined by nonlinear regression using the GraphPad Prism software.

2.3 Crystallization, structure refinement, and analysis The inactivate ZHD101 mutant S102A and the double mutant S102A/V153H were crystallized by using reservoir containing 24% PEG 2000 MME and 0.1 M Bis-Tris


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pH 6.5, similar to the conditions for the wild-type ZHD101.17 A cryoprotectant solution of 28% PEG 2000 MME, 10% glycerol and 0.1 M Bis-Tris, pH 6.5 was used to soak the crystals before flash cooling to 100 K. The S102A/α-ZOL, S102A/V153H/α-ZOL, S102A/β-ZOL and S102A/V153H/β-ZOL complex crystals were obtained by soaking the S102A and S102A/V153H crystals in a cryoprotectant solution that contained 10 mM α-ZOL or β-ZOL for 7 hours.

The diffraction data from these crystals were collected at beam line BL13B1 of the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan), and integrated and scaled by using program HKL2000.18 The four complex structures were found isomorphous to the previous ZHD101 structures (PDB 3WZL and 3WZM). All the structures were refined with non-crystallographic symmetry (NCS) restraints by COOT19 and REFMAC5.20 The drawings of protein structures were prepared with PyMol (http://pymol.sourceforge.net/).

3 Results 3.1 Binding modes of ZHD101 to ZEN and ZOLs By soaking crystals of the inactive mutant S102A, we obtained the ZOL complex structures. The hexagonal crystals of S102A/α-ZOL and S102A/β-ZOL are isomorphous to those of apo-ZHD101 and ZEN-complex, which contain three polypeptide chains (denoted monomer A, B and C) in an asymmetric unit. The electron densities of monomer A and B are clear while that of monomer C is weaker.


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The refined model of monomer C showed a higher temperature factor than A and B.17 Therefore, monomer A and B are used for analysis in the present study. The ZOL molecules were modeled unambiguously according to the electron density map in the complex structures (Fig. 2). Each ZOL molecule binds to the deep pocket between the core and cap domains of ZHD101 (Fig. 3). The RMSD between the ZOL-bound S102A structure and ZEN-bound S102A structure are 0.175 Å (α-ZOL) and 0.156 Å (β-ZOL). The RMSD between two ZOL-bound complexes is 0.166 Å. These results indicate that the substrate-binding does not significantly alter the overall protein structure.

A comparison of the bound ligands shows that the dihydroxybenzoate ring of the substrates superimpose very well. It can probably be attributed to the hydrogen bonding (G32 and S103) and T-stacking force (W183) interactions (Fig. 3B). On the other hand, the large non-planar lactone ring is surrounded by non-polar amino acid residues, which provide weaker van der Waals interactions that may allow more diverse conformations of this ring (Figs. 3B, 4C and S1). The most notable variation lies in the position of H242 side chain in monomer A of the α-ZOL complex and both monomers of the β-ZOL complex. It is different from that in the ZEN complex (Figs. 3B and S2). The deviated H242 side chain disrupts the hydrogen bond network formed among the S102-H242-E126 catalytic triad (Figs. 3B and S1). The improper triad conformation might account for the reduced activity of ZHD101 toward ZOLs.


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Superposition of α-ZOL and ZEN molecules in the complex structures shows that the position of the lactone ring of α-ZOL in monomer A is slightly shifted toward the catalytic core domain as compared with that of ZEN (Fig. 4C). As a result, the putative distance between the H242 NE2 atom and the C8’ of lactone ring is reduced from 3.7 Å to 2.9 Å (see Fig. 4C below). Such a close contact might cause repulsive force between the hydrophilic NE2 and aliphatic C8’ and pushed the H242 side chain away. The effect was not observed in monomer B of S102A/α-ZOL complex, in which the α-ZOL lactone ring was farther away from the H242 NE2 atom (Figs. S1 and S2A).

3.2 Mutation design and hydrolytic activity against ZEN and ZOLs As mentioned above, the substrate-binding pocket allows the bound substrates to adopt various conformations of the lactone ring, but the catalytic triad formation could be disturbed. In an attempt to modify the substrate-binding configuration, we engineered the lactone ring-surrounding residues. V153 and V158 were mutated to polar residues to increase interactions with O6’, and M154 and L135 were replaced by aromatic residues to increase stacking forces. The activities of the mutants are shown in Fig. 5A. No significant improvement in ZOL-hydrolysis was observed for these mutants except for V153H. The mutant V153H exhibited 3.7-fold higher activity against α-ZOL comparing to the wild-type enzyme. More importantly, the mutant V153H preserved capability to hydrolyze ZEN (Table 1A and Fig. 5B). Next, saturated mutation was carried out at the V153 site. As shown in Fig. 5C, V153R,


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V153I, V153N, V153M, and V153T were increased in α-ZOL-degrading activity (by 1.08 to 1.39 folds) but none of them is better than V153H.

3.3 Enzyme characteristics of the mutant V153H The mutant V153H possesses a great potential in industrial applications due to its enhanced ability to hydrolyze α-ZOL, thus the enzyme was further characterized in detail. The mutant enzyme showed optimal activity at similar pH value and temperature with the wild-type enzyme (data not shown). Next, when enzyme thermostability was also examined, we found that both the wild-type enzyme and the mutant V153H are vulnerable to heat treatment because no residual activity was detectable after incubation at 60 °C for 1 min. Nevertheless, V153H retained slightly more activity than did the wild type after heating at 50-55 °C for 1 min (Fig. S3). Since an enzyme additive need to endure 70 °C for at least 2 min to survive the pelleting procedure in feed manufacture, further engineering is required to improve the enzyme thermostability.

The enhanced activity could be attributed to the improved substrate binding ability and/or the elevated catalysis efficiency. In order to investigate the characteristics of the enhanced activity of V153H against α-ZOL, the kinetic parameters of the enzyme were measured. The Km and kcat of wild type ZHD101 against α-ZOL are 63.8 µM and 0.384 s-1, respectively. For V153H mutant, the Km and kcat are 173 µM and 1.996 s-1, respectively (Table 1B). According to these data, the V153H mutant shows about


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2.7-fold reduced substrate-binding affinity but along with a 5.2-fold higher turn-over rate. The kinetic constants of V153H against β-ZOL show no obvious differences compared to those of the wild type enzyme, consistent with the above observation that the specific activity against β-ZOL remains similar.

3.4 ZOL-complex structures of V153H To further elucidate the molecular basis of enhanced α-ZOL hydrolyzing activity of the V153H mutant, crystal structures of the double mutant S102A/V153H in complex with α-ZOL and β-ZOL were determined (Table 2). Similar to the previous structures, only monomer A and B were analyzed here. The RMSD between the structures of ZOL-bound S102A and ZOL-bound S102A/V153H enzymes are 0.133 Å (α-ZOL) and 0.100 Å (β-ZOL), suggesting that the V153H mutation does not significantly affect the overall protein structure. The conformation of bound ligands can be clearly delineated according to the electron density map (Fig. 2).

From the S102A/V153H/α-ZOL complex, we found that the NE2 atom of H153 is within hydrogen bonding distance (3.0 Å) to the C6’ OH of the lactone ring. More importantly, the imidazole side chain of H242 in both monomer A and B rotates to a proper position of forming catalytic triad (Figs. 4A and S1). Superimposing ligands in the complex structures demonstrated that the α-ZOL position in S102A/V153H is apparently farther from the triad than that in the S102A enzyme (Fig. 4C). The substrate is relocated to the original position in the S102A/ZEN complex (Fig. 4C). It


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is assumed that the increased space allows the H242 side chain to come back and form a functional catalytic triad.

On the other hand, H153 seems to cause no significant alteration in β-ZOL binding and the H242 side chain of S102A/V153H/β-ZOL complex remains facing outwards and fails to form a hydrogen-bonding network of S102-H242-E126 (Fig. 4B). These structural observations well correlate with the enzymatic assay.

4 Discussion Lactonases have been widely studied since they participate in regulating microbial growth and pathogenesis by inactivating bacterial N-acyl homoserine lactones (AHL).21 Since the substrates ZEN and AHL share little similarity at chemical structures, the catalysis mechanisms and structures of AHL lactonases and ZHD101 are different. In fact, BLASTp searches with ZHD101 sequence only showed ~30% identity in the N-terminal region with α/β-hydrolases. The most similar protein, enol-lactone hydrolase PcaD (PDB code 2XUA), contains an α-helical cap domain to determine the substrate specificity, which is different from the substrate binding mechanism of ZHD101. Since ZHD101 structure is the first of ZEN lactonase and is far more different than between other lactonases, the study of the structural basis of substrate specificity is meaningful in both research and application aspects.

ZHD101 is so far the most well-studied and effective ZEN detoxifying enzyme,


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which possesses a great potential in industrial applications. Unfortunately, ZHD101 exhibits lower activity in hydrolyzing ZOLs, among which α-ZOL is an even more toxic derivative than ZEN. Intriguingly, the only difference between ZEN, α-ZOL and β-ZOL is the functional group on the C6’ atom, that ZEN carries a keto group and the ZOLs a hydroxyl group (Fig. 1). Therefore, how ZHD101 binds to and differentiates between these different substrates is of great interest. Moreover, deciphering the substrate binding pattern may open a door to engineering ZHD101 to enhance its ZOL-hydrolyzing activity.

In the present study, we analyzed the crystal structures of ZHD101 in complex with ZOLs and found that the catalytic triad is, at least partially, disrupted upon binding to ZOLs. Analysis of the bound substrates suggests that the dihydroxylbenozate part of these related compounds superimpose very well to each other, while the lactone ring conformations are diverse. The lactone ring binds to the open side of the substrate-binding groove, which is formed by several non-polar residues and may allow various conformations. For instance, the lactone ring of α-ZOL is found to bind to a more inward position than that of ZEN, and the aliphatic C8’ atom may repel the hydrophilic H242 side-chain imidazole with a hydrophobic force. As a consequence, the imidazole group of H242 is pushed aside through side chain rotation and fails to mediate the S102-H242-E126 hydrogen bond network. This directly disrupts the catalytic triad formation. The C6’-keto group of ZEN renders the lactone ring with higher rigidity, while the C6’-OH group of α-ZOL allows it to have more flexibility.4


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This is evident by the fact that all ZEN molecules in all complex structures take the same conformation but variations are seen in α-ZOL (Fig. S2). This flexibility might cause a larger space requirement for the α-ZOL lactone ring and might be the reason for H242 being repelled.

Conformational change of an amino acid residue upon binding to a different ligand is not an isolated instance. In the redox sensing protein T-Rex, the binding to NAD+ or NADH causes a translocation of an essential phenylalanine residue and hence affects further binding to another NAD+.22 Histidine in the catalytic triad of hydrolyses, notably serine proteases, is believed to undergo conformational changes during catalysis. This conformational rearrangement tunes the pKa of histidine to facilitate its proton acquisition and release. The change of pKa serves as a significant driving force in the catalytic process.23 In a typical triad-catalyzed reaction, the histidine with high pKa first acquires a proton from the serine. Afterwards, to accomplish proton transfer to the peptide nitrogen, the imidazole ring of the histidine makes a slight turn (~20°) and becomes closer to the nitrogen atom, accompanying the lowered pKa of histidine to facilitate the proton release.24 In the present study, the side chain of H242 is rotated by more than 90°, away from both S102 and the carbonyl group, making the proton transfer impossible. Therefore, the histidine deviation observed in ZHD101 is unique and different from the conformational change as described in the traditional hydrolase reactions.


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In order to restore the catalytic triad configuration, we engineered several residues that interact with the substrate. In the mutant V153H, the α-ZOL lactone ring is fixed by a hydrogen bond between the H153 side chain and the C6’ OH atom of α-ZOL. The lactone is thus shifted outward of the pocket to yield a space that accommodates the H242 side chain (Fig. 4C). As a result, H242 is rotated back to the correct orientation to form a functioning catalytic triad. We believe that these results constitute a successful case of structure-based enzyme engineering, through which the substrate-binding residue is modified to improve catalytic activity.

ZHD101 binding to β-ZOL is a different story. The OH group on the C6’ atom of β-ZOL points to the proximal side of the lactone ring plane, in a different direction compared to α-ZOL (Fig. 1), and forms a hydrogen bond with the NE2 atom of the deviated H242 side chain (Fig. 3B). The misplacement of H242 caused by β-ZOL binding is a result of direct interaction with the substrate, and thus a distinct enzyme engineering strategy may be required to improve the ZHD101 activity for β-ZOL.

5 Conclusions In this study, we identified a novel mutant V153H with improved substrate specificity. The engineered V153H was based on crystallographic structural analysis. Its activity towards α-ZOL was increased by 3.7 folds while the activity towards ZEN was also maintained. The reduced flexibility of α-ZOL caused by a newly formed hydrogen bond with H153 is believed to change the conformation of H242 and thus enhance the


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catalytic efficiency in α-ZOL hydrolysis.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The oligonucleotides for site-directed mutagenesis. Complex structures of ZHD101 (monomer B). Substrates in complex structures. Thermostability of WT and V153H.

Acknowledgments This research was supported by the Taiwan Protein Project of Academia Sinica, funded by Ministry of Science and Technology (MOST105-0210-01-12-01), National Natural Science Foundation of China (grants 31500642, 31570130, 31300615, 31400678 and 31470240) and Tianjin Municipal Science and Technology Commission (13ZCZDSY04800). We thank the National Synchrotron Radiation Research Center of Taiwan for beam-time allocation and data collection assistance.


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Yamaguchi, I.; Kimura, M. Appl. Environ. Microbiol. 2007, 73, 1622-1629. (17) Peng, W.; Ko, T. P.; Yang, Y.; Zheng, Y.; Chen, C. C.; Zhu, Z.; Huang, C. H.; Zeng, Y. F.; Huang, J. W.; Wang, H. J.; Liu, J. R.; Guo, R. T. RSC Advances 2014, 4, 62321-62325. (18) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326. (19) Emsley, P.; Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 2004, 60, 2126-2132. (20) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. Acta Crystallogr. D Biol. Crystallogr. 2011, 67, 355-367. (21) Fast, W.; Tipton, P. A. Trends Biochem Sci 2012, 37, 7-14. (22) McLaughlin, K. J.; Strain-Damerell, C. M.; Xie, K.; Brekasis, D.; Soares, A. S.; Paget, M. S.; Kielkopf, C. L. Mol. Cell 2010, 38, 563-575. (23) Hudaky, P.; Perczel, A. Int. J. Quantum. Chem. 2007, 107, 2178-2183. (24) Hudaky, P.; Perczel, A. J. Phys. Chem. A 2004, 108, 6195-6205.


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ACS Catalysis

TABLE Table 1 Specific activities (A) and kinetic data against ZOLs (B) of WT and V153H mutant

A

ZEN （U/mg））

α-ZOL （U/mg））

β-ZOL （U/mg））

WT

0.93

0.42

0.33

V153H

0.92

1.57

0.23

B Enzyme

Substrate

Km (µM)

kcat (s-1)

kcat/Km (s-1·M-1)

α-ZOL

63.8±3.5

0.384±0.008

6.02×103

β-ZOL

20.9±3.5

0.095±0.005

4.54×103

α-ZOL

173.4±5.3

1.996±0.232

1.15×104

β-ZOL

23.5±3.1

0.144±0.006

6.13×103

WT

V153H


ACS Catalysis

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Table 2 Data collection and refinement statistics for ZHD101 complex crystals S102A/α-ZOL

S102A/β-ZOL

S102A/V153H/α-ZOL

S102A/V153H/β-ZOL

PDB code

5IE4

5IE6

5IE5

5IE7

Data collection

13B1

13B1

13B1

13B1

space group

P6122

P6122

P6122

P6122

a [Å]

86.205

86.043

86.164

86.408

b [Å]

86.205

86.043

86.164

86.408

c [Å]

473.247

470.111

471.862

470.449

unit-cell

α /β /γ (°)

90.00/90.00/120

90.00/90.00/120

90.00/90.00/120

90.00/90.00/120

resolution [Å]

25 - 2.80 (2.90 – 2.80)

25 - 2.67 (2.77 – 2.67)

25 - 2.38 (2.46 – 2.38)

25 – 2.5 (2.59 – 2.5)

unique reflections

26370 (2579)

30272 (2902)

42998 (3933)

37570 (3640)

redundancy

6.2 (6.3)

6.0 (5.9)

6.9 (6.9)

3.8 (3.9)

completeness [%]

97.5 (98.9)

98.3 (97.4)

99.1 (93.2)

99.8 (99.5)

average I/σ (I)

36.7 (10.2)

41.5 (10.1)

27.5 (4.5)

19.5 (3.16)

5.3 (16.3)

4.5 (15.3)

5.9 (28.0)

6.5 (35.7)

no. of reflections

25085 (1822)

28626 (2020)

40758 (2885)

35555 (2521)

Rwork (95 % of data)

0.209 (0.284)

0.220 (0.244)

0.214 (0.271)

0.217 (0.259)

Rfree (5 % of data)

0.263 (0.372)

0.279 (0.280)

0.269 (0.309)

0.289 (0.317)

r.m.s.d. bonds [Å]

0.010

0.010

0.010

0.009

r.m.s.d. angles [º]

1.52

1.529

1.499

1.513

most favored [%]

94.4

94.3

95.5

94.3

allowed [%]

5.0

4.9

3.8

5.0

disallowed [%]

0.6

0.8

0.8

0.6

Protein

5092/39.9

5028/44.5

5301/32.9

5034/35.8

Water

209/42.4

228/60.6

301/53.9

384/49.3

Ligand

46/60.9

46/58.1

46/68.5

46/42.1

[a]

R merge [%]

Refinement

dihedral angles

no. of non-H atoms / average B [Å2 ]

Values in parentheses are for the highest resolution shell. a Rmerge = ∑hkl∑i|Ii(hkl)-| / ∑hkl∑iIi(hkl).


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ACS Catalysis

Figure 1 Chemical structure of ZEN and ZOLs. (A) The hydrolytic process of ZEN by ZHD101 is shown with the structures of the substrate (left), intermediate (middle), and product (right). Structures of α-ZOL (B) and β-ZOL (C) are also shown.


ACS Catalysis

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Figure 2 Electron density maps of ZOL substrates and active-site residues in the complex structures. The 2Fo-Fc electron density maps of the ligand and active-site residues are contoured at 1.0 σ (grey) and 2.0 σ (red) levels. ZOLs and the amino acids are presented as thick stick and thin stick models, respectively. The same region in both monomers A and B of each complex structure is viewed in a similar orientation.


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ACS Catalysis

Figure 3 Complex structures of ZHD101 (monomer A). (A) Overall structure of S102A/α-ZOL is presented as a cartoon model. ZEN from S102A/ZEN, α-ZOL from S102A/α-ZOL, and β-ZOL from S102A/β-ZOL, along with the catalytic triad, are presented as thick stick and thin stick models. The color scheme is green for ZEN, cyan for α-ZOL, and magenta for β-ZOL. (B) Stereo view of the superimposed structures of S102A/ZEN, S102A/α-ZOL, and S102A/β-ZOL. The protein model of S102A/ZEN is shown as cartoon. The presentation and color scheme for the ligands and amino acid residues is as described in panel (A). Dash lines indicate distances within 3.5 Å.


ACS Catalysis

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Figure 4 Active site structures of the mutant complexes (monomer A). (A) The α-ZOL in complex with S102A and S102A/V153H are superimposed. (B) The β-ZOL in complex with S102A and S102A/V153H are superimposed. (C) The structures of S102A/ZEN, S102A/α-ZOL, and V153H/α-ZOL are superimposed and shown in stereo. The substrates and protein side chains are presented as thick and thin stick models. The various positions of C8’ are indicated by black dots.


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ACS Catalysis

Figure 5 Catalytic activity of ZHD101 and mutants. (A) Hydrolytic activity of the wild-type (WT) enzyme and mutants against α-ZOL and β-ZOL. The WT activity is defined as 100%. (B) Hydrolytic activity of WT and V153H mutant against ZEN, α-ZOL and β-ZOL. Each is presented as a percentage of the WT activity against ZEN. (C) Activity of WT and various V153 mutants against α-ZOL. The activity of WT is defined as 100%. All the assays were performed in triplicate and the results presented as average ± SD.


ACS Catalysis

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Table of Contents (TOC)


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ACS Catalysis

ChemDraw Professional 15.0. Figure 1 Chemical structure of ZEN and ZOLs. (A) The hydrolytic process of ZEN by ZHD101 is shown with the structures of the substrate (left), intermediate (middle), and product (right). Structures of a-ZOL (B) and β-ZOL (C) are also shown. 109x47mm (300 x 300 DPI)


ACS Catalysis

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PyMOL 1.2r1 . Figure 3 Complex structures of ZHD101 (monomer A). (A) Overall structure of S102A/a-ZOL is presented as a cartoon model. ZEN from S102A/ZEN, a-ZOL from S102A/a-ZOL, and β-ZOL from S102A/β-ZOL, along with the catalytic triad, are presented as thick stick and thin stick models. The color scheme is green for ZEN, cyan for a-ZOL, and magenta for β-ZOL. (B) Stereo view of the superimposed structures of S102A/ZEN, S102A/a-ZOL, and S102A/β-ZOL. The protein model of S102A/ZEN is shown as cartoon. The presentation and color scheme for the ligands and amino acid residues is as described in panel (A). Dash lines indicate distances within 3.5 Å. 209x88mm (300 x 300 DPI)


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ACS Catalysis

Office 2013. Figure 4 Catalytic activity of ZHD101 and mutants. (A) Hydrolytic activity of the wild-type (WT) enzyme and mutants against α-ZOL and β-ZOL. The WT activity is defined as 100%. (B) Hydrolytic activity of WT and V153H mutant against ZEN, α-ZOL and β-ZOL. Each is presented as a percentage of the WT activity against ZEN. (C) Activity of WT and various V153 mutants against α-ZOL. The activity of WT is defined as 100%. All the assays were performed in triplicate and the results presented as average ± SD. 209x154mm (300 x 300 DPI)


ACS Catalysis

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PyMOL 1.2r1. Figure 5 Active site structures of the mutant complexes (monomer A). (A) The α-ZOL in complex with S102A and S102A/V153H are superimposed. (B) The β-ZOL in complex with S102A and S102A/V153H are superimposed. (C) The structures of S102A/ZEN, S102A/α-ZOL, and V153H/α-ZOL are superimposed and shown in stereo. The substrates and protein side chains are presented as thick and thin stick models. The various positions of C8’ are indicated by black dots. 209x204mm (300 x 300 DPI)


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Enhanced Î±-Zearalenol Hydrolyzing Activity of a Mycoestrogen

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