Crystal Structure of a Mycoestrogen-Detoxifying Lactonase from

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Crystal structure of a myestrogen-detoxifying lactonase from Rhinocladiella mackenziei: molecular insight into ZHD substrate selectivity Yingying Zheng, Wenting Liu, Chun-Chi Chen, Xiangying Hu, Weidong Liu, Tzu-Ping Ko, Xueke Tang, Hongli Wei, Jian-Wen Huang, and Rey-Ting Guo ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00464 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Catalysis

Crystal Structure of a Mycoestrogen-Detoxifying Lactonase from Rhinocladiella mackenziei: Molecular Insight into ZHD Substrate Selectivity

Yingying Zheng,1,† Wenting Liu,1,2,† Chun-Chi Chen,1,† Xiangying Hu,1,3 Weidong Liu,1 Tzu-Ping Ko,4 Xueke Tang,1,5 Hongli Wei,1 Jian-Wen Huang,1 Rey-Ting Guo1,*

1

Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial

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

College of Biotechnology, Tianjin University of Science and Technology, Tianjin 30

0457, China. 3

School of Biotechnology, Jiangnan University, Wuxi 214122, China.

4

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

5

School of Life Science, University of Science and Technology of China, Anhui

230026, China.

*Correspondence E-mail: [email protected] (RTG). †

These authors contributed equally to this work.

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Abstract Development of potent biocatalysts for enzymatic detoxification of estrogenic mycotoxin zearalenone (ZEN) and its more toxic derivative α-zearalenol (α-ZOL) is of great interests. Here, we report the crystal structures of a ZEN-hydrolyzing enzyme from Rhinocladiella mackenziei (RmZHD), including substrate complexes. A molecular mechanism for the distinct activity of RmZHD in hydrolyzing the structurally similar ZEN and α-ZOL is then proposed. Additionally, structure-based engineering to modify the substrate-binding pocket and improve the RmZHD activity towards α-ZOL is presented. These results expand our scope in understanding the catalytic mechanism of ZHD-family enzymes and are of vital importance in further industrial applications.

Key words mycoestrogen, mycotoxin, grain detoxification, ZHD, crystal structure, Rhinocladiella mackenziei, enzyme-substrate complex


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ACS Catalysis

Introduction Zearalenone (ZEN; 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcylic acid lactone; Scheme 1), an estrogenic mycotoxin from Fusarium species, is widely detected in musty grains. Consumption of ZEN-contaminated cereals leads to reproductive disorders in domestic animals and severe health problems in humans, causing significant economic loss1-4. Apart from chemical and physical methods to remove

ZEN,

employing

biocatalysts

which

operate

detoxification

in

a

substrate-specific manner is a more attractive approach to overcome the mycotoxin contamination. Identification of a novel lactonase ZHD101 from Clonostachys rosea that catalyzes ester bond cleavage of ZEN into non-toxic dihydroxyphenyl product with an open side chain provided a key element for the mycotoxin bio-degradation strategy5 (Scheme 1). Our previous study indicates that ZHD101 adopts a novel α/β-hydrolase fold comprising a core domain and a helical cap domain6. Between the two domains, a deep substrate-binding pocket is formed along with the catalytic triad of S102-H242-E126. The enzyme-substrate interactions mainly involve non-polar contacts.

When ingested, the C6’-keto group on the lactone ring of ZEN can be reduced by gut microbes or intracellular enzymes to yield a more toxic derivative α-zearalenol (α-ZOL; Scheme 1)7, which binds to estrogen receptors 10-20 times stronger and exhibits >90-fold higher estrogenic activity comparing to ZEN8. Notably, α-ZOL is slightly less estrogenic comparing to estrogen (~68%) while ZEN exhibits about 1% estrogenic activity of estrogen9. ZHD101 catalyzes the hydrolytic reaction that detoxifies α-ZOL via the same mechanism of ZEN. Intriguingly, ZHD101 activity against α-ZOL is ~40% lower than against ZEN although the two compounds share a


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very similar chemical structure (Scheme 1). Recently, we reported a complex structure of ZHD101 and α-ZOL and found that the hydroxyl group on C6’ endows the α-ZOL lactone ring with higher flexibility, which causes a repulsive force against the nearby catalytic base H2426. As a result, the H242 side chain was pushed aside and the catalytic triad was disrupted. We then modified an adjacent residue V153 to His, which formed an H-bond to the C6’ OH group and shifted the α-ZOL lactone ring to yield a space for H242 to reconstitute the catalytic triad. This variant V153H showed 3.17-fold α-ZOL hydrolyzing activity.

Traditional approach of isolating mycotoxin-degrading microorganisms and identifying the enzymes is a labor-intense process. Consequently, we performed data-mining in Genbank for more potential ZHD-family members instead. All selected candidates possess conserved catalytic features of ZHD101 including the catalytic triad and oxyanion hole. One hypothetical protein from Cladophialophora bantiana (CbZHD) exhibited 0.688 and 0.121 U/mg specific activity against ZEN and α-ZOL, 73% and 17% comparing to ZHD10110. Another hypothetical protein from Rhinocladiella mackenziei that exhibited ZHD activity shares 63.4% amino acid sequence identity to ZHD101 and was later dubbed RmZHD. To get insights into the molecular mechanism of this new ZHD member, we determined the crystal structures of RmZHD and its complexes with ZEN and α-ZOL.


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ACS Catalysis

Results and Discussion The specific activity of RmZHD against ZEN and α-ZOL are 1.379 ± 0.12 and 0.763 ± 0.07 U/mg, respectively, which are higher than that of ZHD101 by about 1.66- and 1.2-fold (Figure S1). These results indicate RmZHD is more efficient comparing to ZHD101 but both enzymes exhibit lower activity toward α-ZOL than ZEN. Sequence alignment with ZHD101 indicates that the catalytic triad of RmZHD comprises S105-H243-E129 (Figure S2). To prevent the substrate from being hydrolyzed upon complex formation, we produced an inactive mutant (S105A) of recombinant RmZHD for crystallographic study. The overall structure of RmZHD is similar to ZHD101 (Cα RMSD = 0.416 Å) that the cap and core domains are divided by a putative substrate-binding cleft (Figure 1). From sequence alignment, the most variable part in RmZHD and ZHD101 is region 133-164 (Figure S2) in the cap domain, which contains a number of substrate-binding residues (Figure 1). In particular, the β6-α5 loop of RmZHD is shifted inward, closer to the substrate-binding pocket when compared with ZHD101 (Figure 1).

Next, high-resolution complex structures were obtained by soaking the S105A crystals with various substrates (Table 1). The corresponding electron densities of the soaked compounds were clearly seen in the substrate-binding pocket (Figure S3). The same substrate-interacting residues were found in ZEN and α-ZOL complex structures (Figure 2). Most lactone ring-interacting residues in RmZHD (L36, M153, F244, Y160, P135, and L138) provide hydrophobic contacts as in ZHD101. The conserved S106 and G35 provide H-bonds to the carbonyl oxygen, with G35 N serving as the oxyanion hole as previously described6. To the phenyl ring moiety there are more polar contacts: W185 provides T-stacking force and an H-bond; S106 OG provides an


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H-bond to O2; N134 ND2 and P190 O provide H-bonds to O4; I193 and P131 provide hydrophobic interactions (Figure 2). Interestingly, the β6-α5 loop becomes even closer (Cα distance ~2 Å) to the substrate when compared to the apo-form structure (Figure S4A). Superposition of the apo and complex structures indicates that the N134 side chain, located in the center of β6-α5 loop, turns to form an H-bond to O4 of the phenyl moiety (Figure S4A). The corresponding position of N134 is a Leu in ZHD101, which does not undergo conformational change upon substrate binding (Figure S4B). To evaluate the catalytic role of N134, the variants N134L and N134A were tested for activity. As shown in Figure 3, both variants exhibit much lower activity against ZEN and α-ZOL than the wild-type enzyme. These results suggest that the N134 plays an important role in RmZHD catalysis.

Another distinct structural difference between RmZHD and ZHD101 is that the entry to the substrate-binding pocket is covered by Y160 and assumes a “closed” conformation in RmZHD, but it remains open in ZHD101 (Figures 2 and 4A). Among the enzyme-substrate interaction network, the Y160 side chain forms a pi-stacking interaction with the planar C5’-(C6’=O)-C7’ keto group (Figure 4B). To investigate the role of Y160 in RmZHD catalysis, the activity of several Y160 variants was examined. As shown in Figure 3, Y160F remained equally active but Y160A lost about 50% ZEN-hydrolyzing activity, indicating that the phenyl ring-mediated pi-stacking force is critical for RmZHD activity against ZEN. However, a Trp side chain may be too bulky to provide a suitable environment for the catalytic reaction, and thus Y160W showed severely decreased activity. Y160G which does not harbor a side chain and has much more conformational flexibility also showed lower activity, implying an indispensable ZEN interaction with residue 160th.


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ACS Catalysis

On the other hand, the α-ZOL lactone ring carries an OH-group on C6’ and lacks a planar moiety like that in ZEN. The lactone ring stretches and extends towards the Y160 side chain (Figure 4B). As a result, the C6’-OH group and the Y160 side chain is so much closer (< 3.5 Å) that it may have a repulsive effect (Figure 4B). We thus examined the α-ZOL-hydrolyzing activity of each Y160 variant (Figure 3). Similar results were observed except Y160A exhibited more than 70 % increase in α-ZOL hydrolyzing activity when compared with the wild type. The striking results of Y160A encouraged us to solve the complex structures of the RmZHD double mutant S105A-Y160A (DM, refinement statistics in Table 1; substrate electron densities in Figure S3). The complex structures indicate that the α-ZOL-binding pattern remains the same as that in the inactive mutant S105A, but the unfavorable close contact caused by Y160 is not observed in S105A-Y160A (Figure S5A). It is likely that the Y160A mutation makes the active-site environment suitable for α-ZOL binding and hydrolysis (Figure S5). On the other hand, Y160A does not form the pi-stacking interaction to the ZEN C5’-(C6’=O)-C7’ plane, which might be attributed to its lower activity in hydrolyzing ZEN (Figure S5B). The active site interaction networks in both complex structures of Y160A mutant remain the same as that seen in the wild type protein (Figure S6).

By using isothermal titration calorimetry (ITC), the dissociation equilibrium constants (KD) of α-ZOL and S105A and S105A/Y160A were determined as 0.44 µM and 0.25 µM (Figure S7). These results suggest that the Y160A mutation increases the α-ZOL binding affinity of the enzyme. Interestingly, the KD of α-ZOL and S105A/Y160G which exhibits very low activity is similar to that of S105A/Y160A


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(0.23 µM). From our previous study, the flexible lactone ring of α-ZOL is mobile upon binding to the active site and may disrupt the catalytic triad formation11. Therefore, although the binding affinity of α-ZOL to Y160G was increased due to removal of the bulky Y160 side chain, certain contact needs to be provided by the 160th amino acid, or the flexible lactone ring may cause unexpected conformational variations in the active site.

In conclusion, we report a novel ZHD which exhibits potent ZEN-hydrolyzing activity for further applications. Its higher efficacy might be attributed to additional substrate interactions via the N134-mediated hydrophilic contacts and Y160-mediated π-stacking. Intriguingly, binding to the even-more-toxic α-ZOL does not result in a favorable configuration in RmZHD due to the close contact between the extended lactone ring and the Y160 side chain. Replacing Y160 by a smaller Ala residue significantly increases the α-ZOL hydrolyzing activity by more than 70%. The corresponding crystal structure indicates that the Ala variant makes a sufficient space to accommodate α-ZOL. These results provide important insights into the molecular mechanism of substrate binding and catalysis for the ZHD-family enzymes, and shall further benefit protein engineering and crop genetic modification to eliminate the mycotoxin.


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ACS Catalysis

Table Table 1. Data collection and refinement statistics for RmZHD 5XO6

S105A /α-ZOL 5XO7

S105A /ZEN 5XO8

5Z5J

DM /α-ZOL 5Z7J

P1

P1

P1

P1

P1

P21

a [Å]

76.01

75.46

75.47

75.38

75.34

44.32

b [Å]

96.88

95.16

95.20

94.97

94.76

110.52

c [Å]

101.90

104.45

101.67

100.50

100.45

127.83

89.0/87.7 /88.0 25-2.38 (2.46-) 114582 (11248)

91.2/92.1 /91.6 25-1.88 (1.95-) 222958 (21671)

90.2/92.3 /91.5 25-1.88 (1.95-) 224832 (22134)

90.7/92.1 /91.9 25-2.18 (2.26-) 143209 (13921)

90.7/92.4 /91.6 25-1.98 (2.05-) 188099 (18576)

90 /100.1 /90 25-2.32 (2.40-) 50048 (5058)

3.9 (3.8)

3.9 (3.7)

4.0 (3.9)

3.4 (3.1)

3.9 (3.7)

3.9 (3.8)

completeness[%]

98.5 (96.9)

97.6 (94.9)

97.8 (96.5)

96.5 (94.1)

97.7 (96.5)

95.0 (96.6)

average I/σ

23.3 (2.5)

18.8 (2.1)

23.4 (3.9)

13.2 (2.4)

9.5 (2.2)

18.1 (3.3)

Rmerge[b] [%]

5.8 (37)

8 (49.8)

5.8 (30.2)

7.3 (48.2)

7.3 (66)

6.7 (48.6)

(92.7)

(82.5)

(94.4)

(80.2)

(87.1)

(80.3)

108546 (8192)

213237 (16246)

211451 (15717)

143070 (11708)

188061 (18019)

47536 (3639)

Rwork (95 % of data)

17.1(26.4)

16.5 (21.7)

16.4 (24.6)

17 (23.1)

16.5 (24.4)

20.1 (29.2)

Rfree (5 % of data)

20.9 (29.1)

19 (24.9)

18.8 (26.3)

21.1 (29.4)

20.9 (28.6)

26 (34.2)

r.m.s.d. bonds [Å]

0.009

0.012

0.011

0.008

0.008

0.010

r.m.s.d. angles [º]

1.2

1.3

1.7

0.9

1.0

1.2

most favored [%]

96.3

97.3

97.2

97.0

96.9

95.9

allowed [%]

3.3

2.3

2.4

2.6

2.7

3.7

disallowed [%]

0.4

0.4

0.4

0.4

0.4

0.4

16470/54.8

16482/27

16447/30.4

16359/36.4

16384/35.4

8174/48.9

water

607/47.3

2241/39.2

2672/46.3

1396/40.1

1864/41.8

250/39.8

ligand

32/67.4

184/38.1

184/40.9

7/39.6

198/43.9

74/44.1

S105A PDB code

DMa

DM/ZEN 5Z97

Data co1lection space group unit-cell

α /β /γ (°)

resolution [Å] unique reflections redundancy

CC1/2 [%] Refinement no. of reflections

dihedral angles

no. of non-H atoms/average B [Å2] protein

Values in parentheses are for the highest resolution shell. a DM, S105A/Y160A double mutant b Rmerge = ∑hkl∑i|Ii(hkl)-| / ∑hkl∑iIi(hkl).


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Supporting Information Statement Experimental methods Table S1. The oligonucleotides for site-directed mutagenesis Figure S1. Hydrolytic activity of ZHD101 and RmZHD. Figure S2. Protein sequence alignment of RmZHD and ZHD101 Figure S3. Electron density maps of ligands in complex structures. Figure S4. The β6-α5 loop conformation in ZHD structures. Figure S5. Substrate-enzyme interaction networks of RmZHD Y160A variant. Figure S6. Substrate-enzyme interaction networks of wild type and Y160A RmZHD. Figure S7. ITC results of wild type and mutant RmZHD binding to α-ZOL.

Acknowledgments This work was supported by the National Natural Science Foundation of China (grants 31470240 and 31570130); CAS Interdisciplinary Innovation Team; Youth Innovation Promotion Association, CAS; Taiwan Young Visiting Scholar Funding, CAS; and by the

Taiwan

Protein

Project

(grants

MOST106-0210-01-15-04

and

MOST107-0210-01-19-02). We thank Jie Shen (Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) for technical support with ITC experiment. The synchrotron data collection was conducted at beam line TPS-5A, BL13C1 and BL15A1 of NSRRC (National Synchrotron Radiation Research Center, Taiwan).


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ACS Catalysis

References 1.

Escriva, L.; Font, G.; Manyes, L., In vivo toxicity studies of fusarium mycotoxins

in the last decade: a review. Food Chem. Toxicol. 2015, 78, 185-206. 2.

Kowalska, K.; Habrowska-Gorczynska, D. E.; Piastowska-Ciesielska, A. W.,

Zearalenone as an endocrine disruptor in humans. Environ. Toxicol. Pharmacol. 2016, 48, 141-149. 3.

Makela, S.; Davis, V. L.; Tally, W. C.; Korkman, J.; Salo, L.; Vihko, R.; Santti,

R.; Korach, K. S., Dietary estrogens act through estrogen receptor-mediated processes and show no antiestrogenicity in cultured breast cancer cells. Environ. Health Perspect. 1994, 102, 572-8. 4.

Miksicek, R. J., Interaction of naturally occurring nonsteroidal estrogens with

expressed recombinant human estrogen receptor. J. Steroid Biochem. Mol. Biol. 1994, 49, 153-60. 5.

Takahashi-Ando, N.; Kimura, M.; Kakeya, H.; Osada, H.; Yamaguchi, I., A novel

lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning. Biochem. J. 2002, 365, 1-6. 6.

Peng, W.; Ko, T. P.; Yang, Y. Y.; Zheng, Y. Y.; Chen, C. C.; Zhu, Z.; Huang, C.

H.; Zeng, Y. F.; Huang, J. W.; Wang, A. H. J.; Liu, J. R.; Guo, R. T., Crystal structure and substrate-binding mode of the mycoestrogen-detoxifying lactonase ZHD from Clonostachys rosea. Rsc Adv. 2014, 4, 62321-62325. 7.

Zinedine, A.; Soriano, J. M.; Molto, J. C.; Manes, J., Review on the toxicity,


ACS Catalysis 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

occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chem. Toxicol. 2007, 45, 1-18. 8.

Shier, W. T.; Shier, A. C.; Xie, W.; Mirocha, C. J., Structure-activity

relationships for human estrogenic activity in zearalenone mycotoxins. Toxicon 2001, 39, 1435-8. 9. Frizzell, C.; Ndossi, D.; Verhaegen, S.; Dahl, E.; Eriksen, G.; Sorlie, M.; Ropstad, E.; Muller, M.; Elliott, C. T.; Connolly, L., Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis. Toxicol. Lett. 2011, 206, 210-7. 10. Hui, R.; Hu, X.; Liu, W.; Liu, W.; Zheng, Y.; Chen, Y.; Guo, R. T.; Jin, J.; Chen, C. C., Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana. Acta Crystallogr., Sect. F: Struct. Biol. Commun. 2017, 73, 515-519. 11. Xu, Z.; Liu, W.; Chen, C.-C.; Li, Q.; Huang, J.-W.; Ko, T.-P.; Liu, G.; Liu, W.; Peng, W.; Cheng, Y.-S.; Chen, Y.; Jin, J.; Li, H.; Zheng, Y.; Guo, R.-T., Enhanced α-zearalenol hydrolyzing activity of a mycoestrogen-detoxifying lactonase by structure-based engineering. ACS Catal. 2016, 6, 7657-7663.


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ACS Catalysis

Scheme 1. (A) The hydrolytic process of ZEN by ZHD. The structures of ZEN (left), hydrolytic intermediate (middle), and product (right) are shown. (B) Structure of α-ZOL. The carbon atoms are numbered 1-6 in the phenyl ring and 1’-12’ in the lactone ring.


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Figures

Figure 1. Overall crystal structure of RmZHD. Front and side view of superimposed RmZHD (blue cartoon model) and ZHD101 structures (orange cartoon model). The cap and core domains of RmZHD and ZHD101 are in deeper and lighter colors, respectively.


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ACS Catalysis

Figure 2. Substrate-enzyme interaction networks of RmZHD. The stereo view of enzyme-substrate interaction networks in (A) S105A/ZEN and (B) S105A/α-ZOL are shown. The protein and substrates are shown in cartoon and stick models, respectively. Orange labels, catalytic triad; black labels, residues providing hydrophobic interactions; magenta labels, residues providing hydrophilic interactions. Dash lines indicate distances within 3.5 Å.


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Figure 3. Mutagenesis experiment. Enzymatic activity of RmZHD mutants against ZEN and α-ZOL was measured and the relative activities of each sample are presented as a percentage of wild type enzyme. The wild type enzyme activity towards ZEN and α-ZOL are presented as 100 %. All assays were performed in triplicate and the results presented as average ± SD.


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ACS Catalysis

Figure 4. Interactions between substrates and Y160. (A) Entry of the substrate-binding pocket of ZHD101/ZEN (left panel) and RmZHD/ZEN (right panel). The overall structures are shown in cartoon models and protein surfaces are displayed. The bound ZEN molecules, RmZHD Y160 and corresponding residue in ZHD101 (V158) are in stick models. (B) Interactions between ZEN or α-ZOL and Y160 in RmZHD complex structures. Some carbons numbering of the lactone ring are shown. Dash lines, distance < 3.5Å.


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Crystal Structure of a Mycoestrogen-Detoxifying Lactonase from

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