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Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the α/β-fold epoxide hydrolase Alp1U

2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês

10.1074/jbc.ra120.015563

ISSN

1083-351X

Autores

Liping Zhang, Bidhan Chandra De, Wenjun Zhang, Attila Mándi, Zhuangjie Fang, Chunfang Yang, Yiguang Zhu, Tibor Kurtán, Changsheng Zhang,

Tópico(s)

Pharmacogenetics and Drug Metabolism

Resumo

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases. Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases. Stereoselective hydrolytic opening of epoxide rings is attractive in asymmetric synthesis, because the resultant vicinal diols are valuable building blocks of chiral drugs and bioactive compounds (1Thibodeaux C.J. Chang W.C. Liu H.W. Enzymatic chemistry of cyclopropane, epoxide, and aziridine biosynthesis.Chem. 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Mutation of tyrosine residues involved in the alkylation half reaction of epoxide hydrolase from Agrobacterium radiobacter AD1 results in improved enantioselectivity.J. Am. Chem. Soc. 1999; 121: 7417-741810.1021/ja990501oCrossref Scopus (65) Google Scholar, 8Yamada T. Morisseau C. Maxwell J.E. Argiriadi M.A. Christianson D.W. Hammock B.D. Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase.J. Biol. Chem. 2000; 275 (10806198): 23082-2308810.1074/jbc.M001464200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 9Hopmann K.H. Himo F. Insights into the reaction mechanism of soluble epoxide hydrolase from theoretical active site mutants.J. Phys. Chem. B. 2006; 110 (17048959): 21299-2131010.1021/jp063830tCrossref PubMed Scopus (41) Google Scholar). Most EHs belong to members of a family with an α/β hydrolase-fold, sharing a catalytic core domain and a cap domain (10Widersten M. Gurell A. Lindberg D. Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.Biochim. Biophys. Acta. 2010; 1800 (19948209): 316-32610.1016/j.bbagen.2009.11.014Crossref PubMed Scopus (71) Google Scholar). The α/β-EHs usually feature a catalytic triad and an oxirane oxygen hole that enable a two-step mechanism involving an SN2 reaction and ester hydrolysis (Fig. 1A) (10Widersten M. Gurell A. Lindberg D. Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.Biochim. Biophys. Acta. 2010; 1800 (19948209): 316-32610.1016/j.bbagen.2009.11.014Crossref PubMed Scopus (71) Google Scholar, 11Nestl B.M. Hammer S.C. Nebel B.A. Hauer B. New generation of biocatalysts for organic synthesis.Angew. Chem. Int. Ed. Engl. 2014; 53 (24520044): 3070-309510.1002/anie.201302195Crossref PubMed Scopus (258) Google Scholar). The catalytic triad contains two aspartates and one histidine (10Widersten M. Gurell A. Lindberg D. Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.Biochim. Biophys. Acta. 2010; 1800 (19948209): 316-32610.1016/j.bbagen.2009.11.014Crossref PubMed Scopus (71) Google Scholar). In the first step, the attack of the nucleophilic aspartate ion against the epoxide ring forms a covalently linked enzyme-acyloxy-substrate adduct (5Borhan B. Jones A.D. Pinot F. Grant D.F. Kurth M.J. Hammock B.D. Mechanism of soluble epoxide hydrolase: formation of an α-hydroxy ester-enzyme intermediate through Asp-333.J. Biol. Chem. 1995; 270 (7592938): 26923-2693010.1074/jbc.270.45.26923Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1: an enzyme to detoxify harmful epoxides.J. Biol. Chem. 1999; 274 (10329649): 14579-1458610.1074/jbc.274.21.14579Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In the second step, the His and the other Asp activate a water molecule to hydrolyze the enzyme-acyloxy-substrate intermediate and release the products as trans-vicinal diols (12Hvorecny K.L. Bahl C.D. Kitamura S. Lee K.S.S. Hammock B.D. Morisseau C. Madden D.R. Active-site flexibility and substrate specificity in a bacterial virulence factor: crystallographic snapshots of an epoxide hydrolase.Structure. 2017; 25 (28392259): 697-707.e410.1016/j.str.2017.03.002Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 13Kong X.D. Yuan S. Li L. Chen S. Xu J.H. Zhou J. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25331869): 15717-1572210.1073/pnas.1404915111Crossref PubMed Scopus (59) Google Scholar). The oxirane oxygen hole contains a pair of tyrosines, which are proposed to donate hydrogen bonds to the oxirane oxygen, thereby assisting the first step of reaction sequence by activating the epoxide and stabilizing the formed oxyanion (6Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1: an enzyme to detoxify harmful epoxides.J. Biol. Chem. 1999; 274 (10329649): 14579-1458610.1074/jbc.274.21.14579Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Amrein B.A. Bauer P. Duarte F. Janfalk Carlsson A. Naworyta A. Mowbray S.L. Widersten M. Kamerlin S.C. Expanding the catalytic triad in epoxide hydrolases and related enzymes.ACS Catal. 2015; 5 (26527505): 5702-571310.1021/acscatal.5b01639Crossref PubMed Scopus (31) Google Scholar). Another family of EHs, represented by the limonene-1,2-epoxide hydrolases, catalyzes a one-step mechanism with SN2 ring opening of the epoxide ring by an enzyme-activated water molecule (Fig. 1B) (15Sun Z.T. Wu L. Bocola M. Chan H.C.S. Lonsdale R. Kong X.D. Yuan S.G. Zhou J.H. Reetz M.T. Structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations.J. Am. Chem. Soc. 2018; 140 (29232125): 310-31810.1021/jacs.7b10278Crossref PubMed Scopus (36) Google Scholar). These EH-catalyzed epoxide ring–opening mechanisms generally follow the SN2 type, with backside attack by the nucleophile resulting in inversion of stereoconfiguration at the reactive carbon center (6Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1: an enzyme to detoxify harmful epoxides.J. Biol. Chem. 1999; 274 (10329649): 14579-1458610.1074/jbc.274.21.14579Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Sun Z.T. Wu L. Bocola M. Chan H.C.S. Lonsdale R. Kong X.D. Yuan S.G. Zhou J.H. Reetz M.T. Structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations.J. Am. Chem. Soc. 2018; 140 (29232125): 310-31810.1021/jacs.7b10278Crossref PubMed Scopus (36) Google Scholar). However, engineering control of the regioselectivity into the epoxide ring–opening reaction remains challenging for EHs (15Sun Z.T. Wu L. Bocola M. Chan H.C.S. Lonsdale R. Kong X.D. Yuan S.G. Zhou J.H. Reetz M.T. Structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations.J. Am. Chem. Soc. 2018; 140 (29232125): 310-31810.1021/jacs.7b10278Crossref PubMed Scopus (36) Google Scholar, 16Li F.L. Kong X.D. Chen Q. Zheng Y.C. Xu Q. Chen F.F. Fan L.Q. Lin G.Q. Zhou J.H. Yu H.L. Xu J.H. Regioselectivity engineering of epoxide hydrolase: near-perfect enantioconvergence through a single site mutation.ACS Catal. 2018; 8: 8314-831710.1021/acscatal.8b02622Crossref Scopus (26) Google Scholar). In synthetic epoxide hydrolysis, the regioselectivity depends on the substituents on the epoxide carbons (17Carlsson A.J. Bauer P. Ma H. Widersten M. Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity.Biochemistry. 2012; 51 (22931287): 7627-763710.1021/bi3007725Crossref PubMed Scopus (35) Google Scholar) and the strength of the nucleophilic reagent. However, in the EH-catalyzed epoxide ring–opening reactions, the regioselectivity is influenced more by the structure of the enzyme active site (18Faber K. Orru R.V.A. Hydrolysis of epoxides.in: Drauz K. Waldmann H. Enzyme Catalysis in Organic Synthesis. Wiley-VCH Verlag GmbH, Weinheim, Germany2002: 579-608Crossref Scopus (5) Google Scholar). These steric constraints can potentially also control the stereochemistry of the product. This hypothesis is supported by enzyme engineering of EHs by directed evolution strategy or small mutagenic library screening (15Sun Z.T. Wu L. Bocola M. Chan H.C.S. Lonsdale R. Kong X.D. Yuan S.G. Zhou J.H. Reetz M.T. Structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations.J. Am. Chem. Soc. 2018; 140 (29232125): 310-31810.1021/jacs.7b10278Crossref PubMed Scopus (36) Google Scholar, 16Li F.L. Kong X.D. Chen Q. Zheng Y.C. Xu Q. Chen F.F. Fan L.Q. Lin G.Q. Zhou J.H. Yu H.L. Xu J.H. Regioselectivity engineering of epoxide hydrolase: near-perfect enantioconvergence through a single site mutation.ACS Catal. 2018; 8: 8314-831710.1021/acscatal.8b02622Crossref Scopus (26) Google Scholar, 19Li C. Kan T.T. Hu D. Wang T.T. Su Y.J. Zhang C. Chen J.Q. Wu M.C. Improving the activity and enantioselectivity of PvEH1, a Phaseolus vulgaris epoxide hydrolase, for o-methylphenyl glycidyl ether by multiple site-directed mutagenesis on the basis of rational design.Mol. Catal. 2019; 47611051710.1016/j.mcat.2019.110517Crossref Scopus (6) Google Scholar, 20Hu D. Zong X.C. Xue F. Li C. Hu B.C. Wu M.C. Manipulating regioselectivity of an epoxide hydrolase for single enzymatic synthesis of (R)-1,2-diols from racemic epoxides.Chem. Commun. 2020; 56 (32030396): 2799-280210.1039/d0cc00283fCrossref PubMed Google Scholar, 21Sun Z. Lonsdale R. Kong X.D. Xu J.H. Zhou J. Reetz M.T. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.Angew. Chem. Int. Ed. Engl. 2015; 54 (25891639): 12410-1241510.1002/anie.201501809Crossref PubMed Scopus (92) Google Scholar). Unfortunately, mechanistic details of what causes regioselectivity improvement of engineered EHs are still elusive. In the biosynthesis of vicinal diols, the chirality of these functional natural products derived from oxiranes plays important roles toward directing of postmodifications of the hydroxy groups, such as acylation and glycosylation. Representative examples include the EH Nasvi-EH1 involved in the biosynthesis of an insect sex attractant (22Abdel-Latief M. Garbe L.A. Koch M. Ruther J. An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site.Proc. Natl. Acad. Sci. U. S. A. 2008; 105 (18579785): 8914-891910.1073/pnas.0801559105Crossref PubMed Scopus (46) Google Scholar), the α/β-EHs NcsF2 and SgcF-catalyzed epoxide ring opening in the biosynthesis of enediyne antitumor antibiotics neocarzinostatins and C-1027 (23Lin S. Horsman G.P. Chen Y. Li W. Shen B. Characterization of the SgcF epoxide hydrolase supporting an (R)-vicinal diol intermediate for enediyne antitumor antibiotic C-1027 biosynthesis.J. Am. Chem. Soc. 2009; 131 (19856960): 16410-1641710.1021/ja901242sCrossref PubMed Scopus (22) Google Scholar, 24Lin S. Horsman G.P. Shen B. Characterization of the epoxide hydrolase NcsF2 from the neocarzinostatin biosynthetic gene cluster.Org. Lett. 2010; 12 (20704329): 3816-381910.1021/ol101473tCrossref PubMed Scopus (15) Google Scholar), the TsrI-mediated endopeptidyl hydrolysis and epoxide ring opening/macrocyclization in thiostrepton biosynthesis (25Zheng Q. Wang S. Duan P. Liao R. Chen D. Liu W. An alpha/beta-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization.Proc. Natl. Acad. Sci. U. S. A. 2016; 113 (27911800): 14318-1432310.1073/pnas.1612607113Crossref PubMed Scopus (23) Google Scholar), and the Alp1U-catalyzed epoxide hydrolysis of epoxykinamycins (Fig. 1C) (26Wang B. Guo F. Ren J.W. Ai G.M. Aigle B. Fan K.Q. Yang K.Q. Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis.Nat. Commun. 2015; 6 (26134788)767410.1038/ncomms8674Crossref PubMed Scopus (29) Google Scholar). Interestingly, we have shown that the Alp1U found in Streptomyces ambofaciens also uses fluostatin (FST) C (1) from Micromonospora rosaria (27Baur S. Niehaus J. Karagouni A.D. Katsifas E.A. Chalkou K. Meintanis C. Jones A.L. Goodfellow M. Ward A.C. Beil W. Schneider K. Süssmuth R.D. Fiedler H.P. Fluostatins C-E, novel members of the fluostatin family produced by Streptomyces strain Acta 1383.J. Antibiot. 2006; 59 (16883779): 293-29710.1038/ja.2006.41Crossref PubMed Scopus (32) Google Scholar, 28Zhang W. Liu Z. Li S. Lu Y. Chen Y. Zhang H. Zhang G. Zhu Y. Zhang G. Zhang W. Liu J. Zhang C. Fluostatins I-K from the South China Sea-derived micromonospora rosaria SCSIO N160.J. Nat. Prod. 2012; 75 (23136829): 1937-194310.1021/np300505yCrossref PubMed Scopus (53) Google Scholar) as a substrate and catalyzes the epoxide hydrolysis to produce a pair of stereoisomers, FST C1 (1a) and FST C2 (1b). The origins of these stereoisomers were proposed to result from nonregioselective hydrolysis by nucleophilic attack at the C-2 and C-3 position of 1 (Fig. 1C) (29Huang C. Yang C. Zhang W. Zhang L. De B.C. Zhu Y. Jiang X. Fang C. Zhang Q. Yuan C.S. Liu H.W. Zhang C. Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines.Nat. Commun. 2018; 9 (29802272)208810.1038/s41467-018-04487-zCrossref PubMed Scopus (38) Google Scholar). Herein, we determined the absolute stereochemistry of the two products FSTs C1 (1a) and C2 (1b) from Alp1U-catalyzed epoxide ring opening of FST C (1) and solved the X-ray crystal structure of Alp1U. An atypical oxirane oxygen hole is identified in Alp1U, consisting of three residues (Trp-186/Trp-187/Tyr-247) that are distinct from the Tyr/Tyr pair in classic α/β-EHs. The regioselectivity was improved for the epoxide ring opening of 1 in the single mutant Y247F to occur predominantly at C-3 and in the double mutant W187F/Y247F mainly at C-2. Single-crystal X-ray structures of these two Alp1U variants complexed with 1 were determined to help infer mechanistic insights into their opposite regioselectivities. We have previously shown that Alp1U converts fluostatin C (1) to two stereoisomers, 1a and 1b, the structures of which have not yet been elucidated (29Huang C. Yang C. Zhang W. Zhang L. De B.C. Zhu Y. Jiang X. Fang C. Zhang Q. Yuan C.S. Liu H.W. Zhang C. Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines.Nat. Commun. 2018; 9 (29802272)208810.1038/s41467-018-04487-zCrossref PubMed Scopus (38) Google Scholar). Subsequently, compounds 1a and 1b were isolated from a large-scale enzymatic reaction with Alp1U and 1 to elucidate the structure. Both 1a and 1b had a molecular formula of C18H14O7 (m/z 341.0672 [M − H]− for 1a, m/z 341.0673 [M − H]− for 1b, calcd. for 341.0667; Fig. S1). The NMR data for 1a and 1b (Fig. 2 and Figs. S1 and S2) showed high similarity to those of the previously characterized FST C (1) (28Zhang W. Liu Z. Li S. Lu Y. Chen Y. Zhang H. Zhang G. Zhu Y. Zhang G. Zhang W. Liu J. Zhang C. Fluostatins I-K from the South China Sea-derived micromonospora rosaria SCSIO N160.J. Nat. Prod. 2012; 75 (23136829): 1937-194310.1021/np300505yCrossref PubMed Scopus (53) Google Scholar). The relatively more downfield shifts of the C-2 and C-3 (Table S1) in 1a and 1b, compared with the same carbons of 1, suggested the presence of a vicinal diol subunit at C-2/C-3 in both compounds, instead of the epoxide moiety at C-2/C-3 in 1 (28Zhang W. Liu Z. Li S. Lu Y. Chen Y. Zhang H. Zhang G. Zhu Y. Zhang G. Zhang W. Liu J. Zhang C. Fluostatins I-K from the South China Sea-derived micromonospora rosaria SCSIO N160.J. Nat. Prod. 2012; 75 (23136829): 1937-194310.1021/np300505yCrossref PubMed Scopus (53) Google Scholar). The trans relative configuration of H-1 and CH3-3 in 1a was indicated by the NOESY correlation of OH-1 and H3-12 (Fig. 2A). To determine the relative configuration of H-1 and H-2, a propylidene acetal derivative of 1a (1a-At) was prepared (Fig. 2A). The cis relative configuration of the 1,2-diol moiety in 1a-At was established by the markedly different proton chemical shifts of the isopropylidene methyl groups (ΔδH 0.36) and the relatively small coupling constant between H-1 and H-2 (3JH-1,H-2 = 5.5 Hz) (Fig. 2A, Fig. S3, and Table S2) (30Hoshino S. Okada M. Wakimoto T. Zhang H. Hayashi F. Onaka H. Abe I. Niizalactams A–C, multicyclic macrolactams isolated from combined culture of Streptomyces with mycolic acid-containing bacterium.J. Nat. Prod. 2015; 78 (26624939): 3011-301710.1021/acs.jnatprod.5b00804Crossref PubMed Scopus (49) Google Scholar, 31Nagle D.G. Gerwick W.H. Structure and stereochemistry of constanolactones A-G, lactonized cyclopropyl oxylipins from the red marine alga Constantinea simplex.J. Org. Chem. 1994; 59: 7227-723710.1021/jo00103a012Crossref Scopus (74) Google Scholar). The NOESY correlation of H-2/OH-3 in 1a-At confirmed the relative trans configuration of OH-2 and OH-3 (Fig. 2A and Fig. S3). Therefore, the relative configuration of the 1,2,3-triol moiety in 1a was determined as (1R*,2R*,3S*) in accordance with the configurations of 1a-At. Considering that 1a was a ring-opened product of 2,3-epoxide 1, it thus must preserve the original (R) absolute configuration at C-1, so the absolute configuration of 1a was assigned as (1R, 2R,3S). The trans relative configuration of H-1 and H-2 in 1b was indicated by the NOESY correlation of H-1 and OH-2 and the large value of the 3JH-1, H-2 coupling constant in methanol-d4 (Table S3 and Fig. S4). The NOESY correlation of H-1 and H3-12 was also observed in 1b (Fig. 2B and Fig. S2), suggesting the cis axial orientation of H-1 and CH3-3 and a trans-vicinal diol moiety at C-2/C-3 in 1b (Fig. 2B). In accordance, the three hydroxyl groups adopted equatorial arrangement in the low-energy computed ωB97X/TZVP polarizable continuum model (PCM)/MeCN conformers of (1R,2S,3R)-1b, whereas H-1 and CH3-3 were axial. The absolute configuration of 1b was determined as (1R,2S,3R) based on the good agreement between the experimental and calculated electronic circular dichroism (ECD) spectra of (1R,2S,3R)-1b (Fig. 2B). To further confirm the absolute configuration of 1b, we tried derivatization with p-bromobenzoyl that was reported to enhance crystallization (32Sy-Cordero A.A. Day C.S. Oberlies N.H. Absolute configuration of isosilybin A by X-ray crystallography of the heavy atom analogue 7-(4-bromobenzoyl)isosilybin A.J. Nat. Prod. 2012; 75 (23116206): 1879-188110.1021/np3005369Crossref PubMed Scopus (17) Google Scholar). A pyridine derivative of 1b (1b-Py) could be obtained as single crystals from the reaction system, and it was determined to have a (3R) configuration by X-ray diffraction analysis, with a Flack parameter of 0.04 (10Widersten M. Gurell A. Lindberg D. Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.Biochim. Biophys. Acta. 2010; 1800 (19948209): 316-32610.1016/j.bbagen.2009.11.014Crossref PubMed Scopus (71) Google Scholar) (Fig. 2C, Fig. S5, and Table S4, CCDC 2016594). This supports the (3R) absolute configuration in 1b. Taken together, the structures of 1a and 1b suggest that the nucleophilic attack by Alp1U at C-2 of 1 produces 1a, whereas 1b derives from the attack at C-3. To learn more details about the catalytic mechanism of the Alp1U reaction, the ligand-free structure of N-terminal His6-tagged Alp1U was determined to 2.45 Å, with four chains per asymmetric unit (Table 1 and Fig. S6). The four Alp1U chains form two dimers according to PISA calculation (33Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372 (17681537): 774-79710.1016/j.jmb.2007.05.022Crossref PubMed Scopus (6771) Google Scholar). Each chain takes the canonical α/β-hydrolase fold, comprising a catalytic domain (residues 27–166 and 251–319) linked to an α-helical cap (residues 167–250) (Fig. 3A). The 26 residues at the N terminus that share no sequence similarity to reported crystal structures of EHs are not resolved in the structure likely because of the flexibility of this region. A PDB database search of the Alp1U coordinates by the Dali server (34Holm L. Benchmarking fold detection by DaliLite v.5.Bioinformatics. 2019; 35 (31263867): 5326-532710.1093/bioinformatics/btz536Crossref PubMed Scopus (212) Google Scholar) reveals that Alp1U is most similar to a fluoroacetate dehalogenase (FAcD, PDB entry 3R3U, Z-score 38.5) (35Chan P.W. Yakunin A.F. Edwards E.A. Pai E.F. Mapping the reaction coordinates of enzymatic defluorination.J. Am. Chem. Soc. 2011; 133 (21510690): 7461-746810.1021/ja200277dCrossref PubMed Scopus (55) Google Scholar). However, Alp1U shares a conserved overall fold (Z-score 38.2–25) with a batch of α/β-EHs, despite low identities in their amino acid sequences (<30%), including the extensively studied epichlorohydrin epoxide hydrolase (EchA) from Agrobacterium radiobacter AD1 (PDB entry 1EHY) (6Nardini M. Ridder I.S. Rozeboom H.J. Kalk K.H. Rink R. Janssen D.B. Dijkstra B.W. The X-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1: an enzyme to detoxify harmful epoxides.J. Biol. Chem. 1999; 274 (10329649): 14579-1458610.1074/jbc.274.21.14579Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), BmEH from Bacillus megaterium ECU1001 (PDB entry 4IO0) (13Kong X.D. Yuan S. Li L. Chen S. Xu J.H. Zhou J. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (25331869): 15717-1572210.1073/pnas.1404915111Crossref PubMed Scopus (59) Google Scholar), the human soluble epoxide hydrolase (sEH, PDB entry 4HAI) (36Pecic S. Pakhomova S. Newcomer M.E. Morisseau C. Hammock B.D. Zhu Z.X. Rinderspacher A. Deng S.X. Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors.Bioorg. Med. Chem. Lett. 2013; 23 (23237835): 417-42110.1016/j.bmcl.2012.11.084Crossref PubMed Scopus (24) Google Scholar), and the bacterial virulence factor EH (cif) from Pseudomonas aeruginosa (PDB entry 5TND) (12Hvorecny K.L. Bahl C.D. Kitamura S. Lee K.S.S. Hammock B.D. Morisseau C. Madden D.R. Active-site flexibility and substrate specificity in a bacterial virulence factor: crystallographic snapshots of an epoxide hydrolase.Structure. 2017; 25 (28392259): 697-707.e410.1016/j.str.2017.03.002Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) (Fig. S7). Structural alignment of Alp1U with these α/β-EHs reveals that the catalytic triad of classic EHs is conserved in Alp1U, consisting of residues Asp-137, His-300, and Asp-161 (Fig. 3B). We propose that Alp1U catalyzes a two-step epoxide ring–opening reaction as the classic α/β-EHs (Fig. 1A). In the first step, Asp-137 launches a nucleophilic attack at the epoxide carbon and forms a covalent alkyl-enzyme intermediate. In the second step, His-300 activates a water molecule, in cooperation with Asp-161, hydrolyzing the alkyl-enzyme intermediate to release the vicinal diol product and Asp-137.Table 1Crystal parameters, data collection, and refinement statisticsAlp1UAlp1UY247F/1Alp1UW187F/Y247F/1PDB ID6KXR6KXH7CLZWavelength (Å)0.978940.978530.97853Resolution range (Å)70.19–2.45 (2.54–2.45)31.98–1.78 (1.84–1.78)44.7–2.1 (2.18–2.1)Space groupP 21 21 21P 21 21 21P 21 21 21Unit cella = 97.09, b = 101.58, c = 117.42, α = β = γ = 90a = 97.48, b = 101.56, c = 117.48, α = β = γ = 90a = 96.66, b = 100.21, c = 117.45, α = β = γ = 90Total reflections86,069 (8505)119,917 (11,484)134,284 (13,302)Unique reflections43,063 (4255)111,803 (10,937)67,180 (6651)Multiplicity2.0 (2.0)1.1 (1.1)2.0 (2.0)Completeness (%)99.35 (99.51)99.88 (99.25)99.98 (99.97)Mean I/σ(I)13.73 (3.21)16.18 (2.99)8.58 (2.95)Wilson B-factor46.6826.4331.22R-merge0.02819 (0.2004)0.112(0.756)0.03037 (0.188)Reflections used for R-free (%)4.774.975.20R-work0.1687 (0.2150)0.1712 (0.2162)0.1932 (0.2190)R-free0.2344 (0.3197)0.2061 (0.2691)0.2468 (0.3006)No. of nonhydrogen atoms923310,0169570Macromolecules909791259078Ligands9117118Water127774354Protein residues117011731172RMSD (bonds) (Å)0.0080.0070.009RMSD (angles) (degrees)1.111.141.22Ramachandran favored (%)969797Ramachandran outliers (%)0.340.340.34Clashscore10.125.817.51Average B-factor45.3034.1032.40Macromolecules45.4033.7032.30Ligands49.5036.8039.30Solvent39.3038.5033.50 Open table in a new tab However, the Alp1U active site is different from those of classic α/β-EHs in the key oxirane oxygen hole (Fig. 3B). The canonical oxirane oxygen hole of α/β-EHs contains two Tyr residues that play critical roles in the first catalytic step, by forming H-bonds with the epoxide oxygen and stabilizing the liberated oxirane oxyanion (Figs. 1A and 3B) (8Yamada T. Morisseau C. Maxwell J.E. Argiriadi M.A. Christianson D.W. Hammock B.D. Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase.J. Biol. Chem. 2000; 275 (10806198): 23082-2308810.1074/jbc.M001464200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Amrein B.A. Bauer P. Duarte F. Janfalk Carlsson A. Naworyta A. Mowbray S.L. Widersten M. Kamerlin S.C. Expanding the catalytic triad in epoxide hydrolases and related enzymes.ACS Catal. 2015; 5 (26527505): 5702-571310.1021/acscatal.5b01639Crossref PubMed Scopus (31) Google Scholar) However, Alp1U has only one of the two conserved tyrosines (Tyr-247), whereas the second Tyr is putatively replaced by a tryptophan (Trp-186) with its indole side chain being positioned for donating an H-bond to the oxirane oxygen (Fig. 3B). Also, the indole side chain of the adjacent Trp-187 is properly positioned to donate an H-bond to the oxirane oxygen (Fig. 3B). Therefore, the three residues Trp-186, Trp-187, and Tyr-247 putatively form an atypical oxirane oxygen hole in Alp1U to donor H-bonds to the epoxide oxygen. To confirm the proposed roles of the catalytic residues, the Alp1U mutants were generated by site-directed mutagenesis (Fig. 3C). As expected, three separate mutants, D137N, H300F, and H300G, completely abolished catalytic activity for 1 (Fig. 3C). The W186F mutant completely lost the activity, whereas the W187F mutant maintained the activity (Fig. 3C). It seems that Trp-186, rather than Trp-187, is required catalytically to donate an H-bond to the oxirane oxygen in 1. Subsequently, the W186Y mutant was made to mimic the classic oxirane oxygen hole in EHs (10Widersten M. Gurell A. Lindberg D. Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis.Biochim. Biophys. Acta. 2010; 1800 (19948209): 316-32610.1016/j.bbagen.2009.11.014Crossref PubMed Scopus (71) Google Scholar). However, W186Y showed no activity (Fig. 3C), likely because the side chain of Tyr-186 could not properly orient to interact with the oxirane oxygen. It should be noted that a histidine (His-115) forms the oxirane oxygen hole with Tyr-219 in the bacterial virulence factor EH cif (Fig. 3B) (37Bahl C.D. Madden D.R. Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair.Protein Pept. Lett. 2012; 19 (21933119): 186-19310.2174/092986612799080392Crossref PubMed Scopus (17) Google Scholar), and a W186H mutant was also constructed. However, the W186H mutant only showed trace catalytic activity for the epoxide ring opening of 1 (Fig. 3C). The conserved Tyr-247 was expected to assist the epoxide ring–opening reaction by donating an H-bond to the oxirane oxygen. Surprisingly, the Y247F mutant retained most activity but selectively produced 1b (98%), preferring a nucleophilic attack at C-3 (Fig. 3, C and D). The substrate conversion rates of W187F and Y247F suggest that neither Trp-187 nor the conserved Tyr-247 in Alp1U is a key H-bond donor to stabilize the oxirane oxygen of 1. To verify this hypothesis, the W187F/Y247F double mutant was constructed. Unexpectedly, the Alp1UW187F/Y247F mutant selectively produced 1a (94%) as a result of preferentially attacking at C-2. These observations are in sharp contrast to previous reports that classic α/β-EHs slightly lost the activity if one of the two catalytic Tyr residues in the oxirane oxygen hole was mutated and became completely inactive if both Tyr residues were mutated (7Rink R. Spelberg J.H.L. Pieters R.J. Kingma J. Nardini M. Kellogg R.M. Dijkstra B.W. Janssen D.B. Mutation of tyrosine residues involved in the alkylation half reaction of epoxide hydrolase from Agrobacterium radiobacter AD1 results in improved enantioselectivity.J. Am. Chem. Soc. 1999; 121: 7417-741810.1021/ja990501oCrossref Scopus (65) Google Scholar). To the best of our knowledge, such significant regioselectivity improvement and switch by mutated residues of the oxirane oxygen hole in Alp1U are unprecedented in EHs that catalyze epoxide ring–opening reactions. To investigate how the Alp1U mutants achieved the regioselectivity, the crystal structures of the Alp1UY247F (1.78 Å) and Alp1UW187F/Y247F (2.1 Å) mutants were determined in complex with the substrate FST C (1), by soaking with an excess of 1 (Table 1). In both crystal structures, the clear electron density of 1 is observed (Fig. 4B and Fig. S8). Superposition reveals that the two structure complexes are essentially identical to the apo-Alp1U structure (Fig. S9). The catalytic cavity of Alp1U forms a deep groove, with a negatively charged surface, which binds the substrate FST C (1) (Fig. 4A). FST C (1) is bound to Alp1U mainly by direct and water-mediated hydrogen bond networks (Fig. 4B). The OH-1 hydroxyl group of 1 orients to the inner side of the active site. Therefore, the O-acyl or O-methyl derivatization at OH-1 could hamper FSTs from entering the Alp1U active site for a steric crowding effect. This might explain our previously reported phenomenon that Alp1U fails to convert FSTs containing an O-acyl or O-methyl group at the C1 position (29Huang C. Yang C. Zhang W. Zhang L. De B.C. Zhu Y. Jiang X. Fang C. Zhang Q. Yuan C.S. Liu H.W. Zhang C. Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines.Nat. Commun. 2018; 9 (29802272)208810.1038/s41467-018-04487-zCrossref PubMed Scopus (38) Google Scholar). Similar binding patterns of 1 are observed in both structures of Alp1UY247F and Alp1UW187F/Y247F in complex with 1 and suggest that Alp1U uses the same epoxide ring–opening mechanism as that of classic α/β-EHs (Fig. 4, C–E). In both structures (Fig. 4, C and D), the carboxyl side chain of Asp-137 locates just below the epoxide ring of 1, ready for the nucleophilic attack. His-300 locates adjacent to Asp-137 and forms a hydrogen bond with Asp-161, consistent with its general role to activate one water molecule for hydrolyzing the covalent substrate-enzyme adduct and the subsequent release of Asp-137. The epoxide ring of FST C (1) is accommodated in the putative oxirane oxygen hole defined by the side chains of Trp-186, Trp-187, and Tyr-247 (Fig. S10). Trp-186 is perfectly positioned to synchronously protonate the epoxide oxygen, consistent with its critical role for the activity. The epoxide oxygen of 1 locates in hydrogen bond distance (Fig. 4F) to the indole N-H of Trp-187 (3.24 Å) and OH of Tyr-247 (2.81 Å). However, the N-H···O angle and O-H···O angle (Fig. 4F) are inappropriate to donate H-bonds, considering that the ideal X-H···Y angle for a hydrogen bond is generally linear or close to 180° (38Shahi A. Arunan E. Why are hydrogen bonds directional?.J. Chem. Sci. 2016; 128: 1571-157710.1007/s12039-016-1156-3Crossref Scopus (23) Google Scholar). This may explain why the W187F and Y247F mutants retain the catalytic activity. Structural alignments of Alp1U, Alp1UY247F/1, and Alp1UW187F/Y247F/1 complexes reveal highly overlapped Asp-137, His-300, Asp-161, and Tyr-247 but a slight displacement of Trp-186 and the adjacent Trp-187 (Fig. 4F). Structural comparison reveals that the substrate 1 in Alp1UW187F/Y247F adopts a ∼20° anticlockwise rotation (Fig. 4F), compared with that in Alp1UY247F. We propose that the substrate rotation is caused by repulsive forces from the two hydrophobic side chains of Phe-247 and Phe-187 in Alp1UW187F/Y247F. This rotation brings the carboxyl oxygen of Asp-137 significantly closer to C-2 of 1 in the Alp1UW187F/Y247F/1 complex (2.91 Å) than in the Alp1UY247F/1 complex (3.18 Å). This new positioning likely directs the regioselective attack at C-2 of the epoxide ring to selectively produce 1a (94%). This observation is consistent with previous studies describing the distances between the nucleophile and epoxide carbons as essential toward determining the regioselectivity of other EHs (14Amrein B.A. Bauer P. Duarte F. Janfalk Carlsson A. Naworyta A. Mowbray S.L. Widersten M. Kamerlin S.C. Expanding the catalytic triad in epoxide hydrolases and related enzymes.ACS Catal. 2015; 5 (26527505): 5702-571310.1021/acscatal.5b01639Crossref PubMed Scopus (31) Google Scholar, 15Sun Z.T. Wu L. Bocola M. Chan H.C.S. Lonsdale R. Kong X.D. Yuan S.G. Zhou J.H. Reetz M.T. Structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations.J. Am. Chem. Soc. 2018; 140 (29232125): 310-31810.1021/jacs.7b10278Crossref PubMed Scopus (36) Google Scholar, 20Hu D. Zong X.C. Xue F. Li C. Hu B.C. Wu M.C. Manipulating regioselectivity of an epoxide hydrolase for single enzymatic synthesis of (R)-1,2-diols from racemic epoxides.Chem. Commun. 2020; 56 (32030396): 2799-280210.1039/d0cc00283fCrossref PubMed Google Scholar). However, the Y247F mutant prefers a regioselective attack at C-3 of 1 to produce 1b (98%), despite the similar distances between the nucleophile Asp-137 and C-2 (3.18 Å) and between Asp-137 and the C-3 carbon (3.24 Å) in the Alp1UY247F/1 complex. It is suggested from the Alp1UY247F/1 complex that the hydrophobic effect of the phenyl side chain, introduced by the Y247F mutation, disfavors the carboxyl anion of Asp-137 to approach C-2 of 1 and largely reduces the preference for attack at C-2. Thus, Alp1UY247F led to a regioselective attack at C-3. The C-3 selectivity of the Y247F mutant suggests that the regioselectivity for epoxide hydrolysis may not be solely determined by the distance of the interacting atoms, especially when the distances to both epoxide carbons are similar. Other parameters, such as hydrophobic effects, must be considered to account for the unexpected regioselectivity. In this work, we identified that Alp1U structurally shows a conserved overall fold as classic α/β-EHs, but it features a unique triad (Trp-186/Trp-187/Tyr-247) to assemble an atypical oxirane oxygen hole that is usually defined by two residues of Tyr/Tyr for the canonical α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U led to improved and switched regioselectivity for the epoxide hydrolysis of 1. The single site mutant Y247F regioselectively attacks at C-3 (98%) of FST C (1Thibodeaux C.J. Chang W.C. Liu H.W. Enzymatic chemistry of cyclopropane, epoxide, and aziridine biosynthesis.Chem. Rev. 2012; 112 (22017381): 1681-170910.1021/cr200073dCrossref PubMed Scopus (187) Google Scholar) to form 1a, the (1R,2R,3S)-isomer, whereas the double mutant W187F/Y247F prefers attacking regioselectively at C-2 (96%) to form the (1R,2S,3R)-1b. Crystal structures of the two mutants with distinctly different regioselectivities in complex with FST C (1) were determined to reveal slight conformation changes of the substrate and residues of the oxirane oxygen hole. These structural and mutagenesis studies of Alp1U and its mutants provided further insight into the factors governing the regioselectivity of α/β-EHs, which suggests that other parameters, such as hydrophobic effects, should be considered, in addition to the distance of the interacting atoms.

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