Subscriber access provided by UNIV OF DURHAM
Article
Mechanistic Insights into the Decoupled Desaturation and Epoxidation Catalyzed by Dioxygenase AsqJ Involved in the Biosynthesis of Quinolone Alkaloids Hao Su, Xiang Sheng, Wenyou Zhu, Guangcai Ma, and Yongjun Liu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01606 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Catalysis is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 22
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
ACS Catalysis
Mechanistic Insights into the Decoupled Desaturation and Epoxidation Catalyzed by Dioxygenase AsqJ Involved in the Biosynthesis of Quinolone Alkaloids
Hao Su, Xiang Sheng, Wenyou Zhu, Guangcai Ma, Yongjun Liu* School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China.
ABSTRACT: AsqJ from Aspergillus nidulans is a non-heme FeII/α-ketoglutarate-dependent dioxygenase that catalyzes the conversion of benzodiazepinedione into 4′-methoxyviridicatin, which is a key step in the biosynthesis of quinolone alkaloids. A series of recent experiments have demonstrated that AsqJ is able to perform the decoupled desaturation and epoxidation reactions. Herein, on the basis of the published crystal structures, combined quantum mechanics and molecular mechanics (QM/MM) calculations have been performed to explore both the desaturation and epoxidation processes. Our calculations reveal that the quintet state of the FeIV–O complex is the ground state, and the catalytic reaction occurs on the quintet state surface. The FeIV-oxo species should firstly undergo an isomerization to initiate the reactions. In the desaturation process, the abstraction of the first hydrogen atom is suggested to follow the σ-channel mechanism. This step is calculated to be rate-limiting with an energy barrier of 19.3 kcal/mol. The abstraction of the second hydrogen atom is found to be quite easy. After the desaturation process, the regenerated FeIV-oxo species firstly attacks the C=C bond of the desaturated intermediate to form a carbon-based radical intermediate, corresponding to an energy barrier of 18.1 kcal/mol, then the radical intermediate completes the ring closure with a barrier of 3.9 kcal/mol. Besides, the calculations using the substrate analogous that lacks the N4-methyl reveal that the H-atom abstraction by FeIV-oxo is still accessible, which suggests that the absence of N4-methyl does not affect the desaturation process itself, but may influence the other processes which are prior to the desaturation. Keywords: QM/MM; nonheme dioxygenase; desaturation; epoxidation; AsqJ.
Corresponding author: E-mail:
[email protected]; Fax: +86 53188564464; 1
ACS Paragon Plus Environment
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
Page 2 of 22
1. Introduction The quinolone scaffold plays an important role in building chemical libraries of bioactive compounds. The
pharmaceutically
attractive
scaffold
of
4′-methoxyviridicatin
(4,
Scheme1),
4-arylquinolin-2(1H)-one, has been found in a variety of quinolone and quinolinone alkaloids [1-3] which exhibit extensive activities of antimicrobial, antimalarial, antiviral and anticancer activities.[4, 5] Thus, understanding the biosynthesis of 4-arylquinolin-2(1H)-one framework is of vital importance for both fundamental enzymology and medicinal purposes.
Scheme 1. Reaction catalyzed by AsqJ enzyme. In 2014, Ishikawa et al. discovered that AsqJ enzyme from Aspergillus nidulans is involved in the biosynthesis
of
4′-methoxyviridicatin.[6]
This
enzyme
belongs
to
the
FeII/α-ketoglutarate
(αKG)-dependent dioxygenase superfamily, which can catalyze diverse reactions including desaturation, epoxidation, halogenation, ring expansion, and hydroxylation, etc.[7-11] Very interestingly, AsqJ is able to catalyze the decoupled desaturation and epoxidation of the 4′-methoxycyclopeptin 1, which is the product
of
the
AsqK-catalyzed
nonribosomal
peptide
synthetase
(NRPS),
to
generate
4′-methoxycyclopenin (3), as shown in Scheme 1. The transformation of 3 to 4 is a non-enzymatic elimination/rearrangement process that is initiated by the final step of epoxidation.[12, 13] In 2016, Brӓuer et al. reported a series of crystal structures of AsqJ from Aspergillus nidulans and mimicked the various stages of the reaction cycle.[14] It was demonstrated that one molecule of dioxygen and αKG are required for both the desaturation and epoxidation processes. The structural analysis of the metal binding center of AsqJ shows the common feature of FeII/αKG dioxygenases, i.e., the iron center coordinates with a 2-His-1-carboxylate facial triad and leaves three binding sites available for the chelation of αKG 2
ACS Paragon Plus Environment
Page 3 of 22
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
ACS Catalysis
and dioxygen molecule.[15] To initiate the catalysis, αKG is firstly oxidized by the FeIII-superoxo intermediate,[16-18] and the generated high valent FeIV-oxo intermediate is considered to be the key species to perform the subsequent oxidation reactions. [11, 19]
Scheme 2. Proposed catalytic cycle of AsqJ. According to the crystal structures and experimental observations, a catalytic picture of AsqJ has been suggested, as shown in Scheme 2.[6, 14] Firstly, the high-spin FeIV-oxo intermediate abstracts a hydrogen atom, either from C3 or C21 of the substrate 1, to generate a substrate radical; then, the FeIII-OH intermediate abstracts the second hydrogen atom of the substrate to complete the desaturation, generating the alkene (Z)-2 intermediate. In the epoxidation process, the regenerated FeIV-oxo attacks one carbon of the C=C bond of 2 to produce the oxirane intermediate 3. Additionally, in vitro activity assays of AsqJ suggested that the N-methyl group of the substrate 1 is essential for catalysis, but the methoxy group of the benzene ring is not required.[14] Recently, Chang et al have experimentally demonstrated that AsqJ uses the FeIV-oxo species to carry out the epoxidation.[20] Although abundant information concerning the catalysis of AsqJ has been derived from the experiments,[6, 14, 20], open questions still remain. For example, on the basis of the crystal structure of AsqJ, the sixth coordination site, which is supposed to be the binding position of dioxygen, is almost perpendicular to the substrate. According to the previous studies of FeII/α-ketoglutarate-dependent dioxygenase superfamily, the generated FeIV-oxo intermediate would occupy this site.[21, 22] However, in AsqJ, OFe is at a distance of ~5.5 Å to the C3 of the substrate, which makes the direct abstraction of C3-H to be unlikely. Thus, a rotation of FeIV-oxo species may be required, as proposed in the studies of 3
ACS Paragon Plus Environment
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
AlkB repair enzymes [23] and carbapenem synthase (CarC)[24]. In addition, some reaction details need clarification, such as which hydrogen atom (H21 or H3) is firstly abstracted by the FeIV-oxo to create the double bond, which carbon firstly forms C–O bond with the OFe, and the structures and energetics of transition states and intermediates are still unknown. Besides, previous studies on P450 enzymes indicated that a radical intermediate is formed upon the attack of FeIV-oxo on the double bond,[25-29] however, recent study on the AlkB enzyme revealed a zwitterion intermediate in the similar process, which can not lead to the epoxidation product by the ring closure mechanism [30]. Thus, the nature of intermediate during the epoxidation is ambiguous. To clarify these issues, on the basis of the recently obtained crystal structures,[14] combined quantum mechanics/molecular mechanics (QM/MM) calculations have been carried out on the AsqJ-catalyzed desaturation and epoxidation reactions. Although some catalytic reactions of other FeII/αKG dioxygenases have been studied by QM/MM methods,[23, 24, 30-32] AsqJ is special for it can perform the stepwise desaturation and epoxidation processes, and a theoretical investigation is still necessary.
2. Computational Details 2.1 MD Simulations Based on the X-ray crystallographic structure of AsqJ (PDB: 5DAQ, Resolution: 1.7 Å),[14] the initial model of enzyme-substrate complex was established. This X-ray structure represents a monomer which includes the natural substrate 4′-methoxycyclopeptin and the co-substrate αKG, whereas the catalytic metal Fe2+ is substituted by a Ni2+, as shown in Figure 1. The substrate 4′-methoxycyclopeptin is located in the vicinity of the metal center, and one water molecule (W) coordinates to the metal. The neighboring residues N157 and Q131 form hydrogen bonds with D136 and αKG. According to the previous studies, after the oxidative decarboxylation of αKG, the generated oxo group will occupy the position of O2. [33, 34] Thus, to construct the model of the reactant FeIV–oxo species (named as Re), we replaced the nickel with iron, and changed the coordinated water molecule and αKG to oxo group and succinate acid, respectively. By comparing the crystal structure of AsqJ with those of two other FeII/αKG dioxygenases (CarC and AlkB in Figure 1), we found that an Arg residue (R267 in CarC or R210 in AlkB) forms hydrogen bond to αKG [23, 35, 24, 36]. This Arg residue is suggested to play important role for the isomerization of FeIV–oxo species. However, in AsqJ (PDB: 5DAQ) this Arg residue is not observed, but a Gln residue 4
ACS Paragon Plus Environment
Page 4 of 22
Page 5 of 22
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
ACS Catalysis
(Q131) is located on the opposite side of the imaginary Arg residue and is within hydrogen bonding distance to αKG. Therefore, the active site of AsqJ is slight different from those of CarC and AlkB, and the formation of key FeIV-oxo may be different accordingly.
Figure 1. Comparison of the active site of AsqJ (5DAQ) with those of CarC (4OJ8) and AlkB (3I2O). Based on the calculation results of pKa from PROPKA server [37, 38] and experimental conditions (pH=7.4), the protonation states of all titratable amino acids were assigned. All Glu and Asp residues were deprotonated while Lys and Arg were protonated, and all His residues were singly protonated according to their pKa values. Then, the missing hydrogen atoms were added by using the HBULID in the CHARMM package.[39] Subsequently, the obtained structure was solved into a pre-equilibrated TIP3P model [40] water sphere with radius of 35 Å and all the added water molecules that locate within 2.8 Å from the heavy protein atoms were removed, and several Na+ ions were randomly added to neutralize the system. Before the molecular dynamics (MD) simulations, a series of energy minimizations were performed using CHARMM program. After that, a spherical boundary potential is employed to prevent water molecules from escaping into the vacuum. Finally, to equilibrate the prepared system, 8 ns MD simulations (MD-I) were performed under stochastic boundary conditions at 298 K. During the MD simulations, the CHARMM22/CMAP all-atom force field [41] was employed. During the MD simulations, the Fe atom as well as the distances of Fe with its coordinated atoms were kept frozen, which is a commonly used strategy for treating the non-heme dioxygenases.[42-44] The calculated root-mean-squared deviations (RMSD) of the protein is shown in Figure S1. One can see that the system is basically equilibrated after 4 ns. Thus, three snapshots were selected from the MD trajectory at 6, 7 and 8 ns as starting models (named as Mn6, Mn7 and Mn8) for the subsequent QM/MM calculations. 2.2 QM/MM Calculations
5
ACS Paragon Plus Environment
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
In QM/MM calculations, the whole system was partitioned into two parts: the QM and MM regions. As shown in Figure 2, the QM region contains the substrate, FeIV-oxo group, the side chains of three Fe-coordinated residues (H134, D136 and H211), succinic acid, and the side chains of residues Q131 and N157 that form hydrogen bonds with succinic acid and D136. The rest of the system was assigned to the MM region. The hydrogen-link atoms with the charge shift model [45, 46] were employed to treat the QM/MM linkages. The electrostatic interaction between QM region and MM region was handled by electrostatic embedding scheme, [47] which incorporates the MM charges into the one-electron Hamiltonian of the QM calculation. All residues and water molecules within 14 Å from the iron center were allowed to relax, while the rest of the system was kept frozen.
Figure 2. Selected QM region for our QM/MM calculations. For geometry optimizations, we employed the unrestricted B3LYP (UB3LYP) [48-50] functional, with the LANL2DZ [51-53] pseudo potential for iron and 6-31G (d, p) for other elements (B1) of the QM region. The MM region was described by the CHARMM22/CMAP force field. The B3LYP functional has been successfully used in the studies of FeII/αKG dioxygenases.[23,24,30-32] Geometry optimizations were done using the hybrid delocalized internal coordinates (HDLCs) optimizer [54], and the limited memory Broyden-Fletcher- Goldfarb-Shanno (L-BFGS) algorithm [55, 56] was used for minima search. To locate the transition states, potential energy profiles along the reaction coordinates were firstly scanned, and the highest points on the profiles were further optimized with the partitioned rational function optimization (P-RFO) algorithm.[57] Based on these optimized geometries, single-point calculations were performed at UB3LYP/CHARMM22/CMAP level with a larger basis set (Wachters all-electron basis set for iron and the 6-31++G (d, p) for the remaining atoms, B2). All
6
ACS Paragon Plus Environment
Page 6 of 22
Page 7 of 22
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
ACS Catalysis
QM/MM calculations were performed with ChemShell package [58], which integrates the TURBOMOLE program [59] and DL-POLY program [60] for QM and MM calculations, respectively.
3. Results and Discussion On the basis of the previously proposed mechanism,[6, 14, 20] the catalytic reaction of AsqJ with 4′-methoxycyclopeptin as substrate was studied by using QM/MM methodology. Our calculation results suggest that an isomerization of the FeIV-oxo species is required before each oxidation process (desaturation and epoxidation). Thus, the whole catalytic reaction can be divided into three sections: the isomerization of FeIV-oxo, the desaturation and epoxidation. In the following subsections, we will discuss these sections separately. 3.1 Isomerization of Iron(Ⅳ Ⅳ)-oxo species The three snapshots (Mn6, Mn7 and Mn8) selected from MD simulations were firstly optimized by QM/MM method, and the optimized structures are denoted as Ren6, Ren7 and Ren8, respectively. On the basis of our calculations, the quintet state is the ground state of FeIV-oxo species, and both the triplet and septet states are higher than the quintet state over 10 kcal/mol (Table S1). It is in line with the previous computational studies of FeIV-oxo species, i.e., the energies of these species follow the spin ordering of quintet
