Theoretical Studies on the Mechanism of Thioesterase-Catalyzed

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Theoretical studies on the Mechanism of Thioesterasecatalyzed Macrocyclization in Erythromycin Biosynthesis Xiong-Ping Chen, Ting Shi, Xiao-Lei Wang, Jitao Wang, Qihua Chen, Linquan Bai, and Yi-Lei Zhao ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01154 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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ACS Catalysis

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Theoretical studies on the Mechanism of

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Thioesterase-catalyzed Macrocyclization in Erythromycin

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Biosynthesis

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Xiong-Ping Chen,1,# Ting Shi,1,# Xiao-Lei Wang,1 Jitao Wang,1 Qihua Chen,1

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Linquan Bai,1 Yi-Lei Zhao1,*

7 8 9 10 11

1

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

12 13 14 15 16 17 18

*To whom correspondence should be addressed:

19 20

Prof. Yi-Lei Zhao

21

School of Life Sciences and Biotechnology

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Shanghai Jiao Tong University

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800 Dongchuan Road, Shanghai 200240, China

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Tel/Fax: +86-21-34207190;

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Email: [email protected]

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ACS Paragon Plus Environment

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TABLE OF CONTENTS (TOC) GRAPHIC

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ABSTRACT

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Macrocyclic polyketides, biosynthesized by modular polyketide synthases (PKSs),

3

have successfully been developed into generation-by-generation pharmaceuticals for

4

numerous therapeutic areas. A great effort has been made experimentally and

5

theoretically to elucidate the biosynthesis mechanisms, in particular for thioesterase

6

(TE)-mediated macrocyclization, which controls the final step in the PKS

7

biosynthesis and determines chemical structures of the final products. To obtain a

8

better insight into the macrocyclization process (i.e. releasing step), we carried out

9

MD

simulations,

QM

and

QM/MM

calculations

on

complexes

of

10

6-deoxyerythronolide B synthase (DEBS) TE and two substrates, one towards a

11

macrocyclic product and another towards a linearly hydrolytic product. Our

12

investigation showed the induced-fit mutual recognition between the TE enzyme and

13

substrates: in the case of macrocyclization a critical hydrogen bonding network is

14

formed between the enzyme and substrate 1, and a hydrophobic pocket appropriately

15

accommodates the substrate in lid region, in which a pivotal pre-reaction state (1IV')

16

with energy barrier of 11.6 kcal/mol was captured on the potential energy surface

17

calculation. Accompanied with the deprotonation of the pre-reaction state, the

18

nucleophilic attack occurs with a calculated barrier of 9.9 kcal/mol and leads to the

19

charged tetrahedral intermediate. Following the decomposition of the intermediate, the

20

final macrocyclic product releases with a relatively low barrier. However, in the case

21

of hydrolysis such a pre-reaction state for cyclization was not observed in similar

22

molecular simulations. These calculations are consistent with the previous biochemical

23

and structural studies about the TE-mediated reactions. Our study indicated that the

24

enzyme-substrate specificity stems from mutual molecular recognition via a

25

pre-reaction

26

pre-reaction-and-action mechanism in the TE macrocyclization and release of PKS

27

product.

28

KEYWORDS

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PKS, thioesterase, macrocyclization, hydrolysis, MD, QM/MM

state

between

DEBS

TE

and

substrates,

3


suggesting

a

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INTRODUCTION

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Modular polyketide synthases (PKSs) are a family of multienzyme complexes,

3

which catalyze numerous macrocyclic polyketides during biosynthesis. Macrocyclic

4

polyketides, possessed of broad and potent biological activities, have developed into

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successful pharmaceuticals such as macrolide antibiotics, antifungal agents,

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immunosuppressants and anticancer agents.1–4 In terms of biosynthesis, type I PKS is a

7

collection of multi-domain assembly lines with multiple requisite domains (namely

8

“modules”), each of which is responsible for a particular function in the construction of

9

polyketides. The 6-deoxyerythronolide B synthase (DEBS), which catalyzes the

10

formation of macrocyclic core of erythromycin, is a prototypical example of type I

11

PKS.5,6 It consists of one initiation module, six elongation modules, and one

12

thioesterase (TE) domain.7 The TE domain is covalently linked to the acyl carrier

13

protein (ACP) domain of the last module, responsible for final cyclization and

14

concomitant release of macrocyclic polyketide. The TE-mediated offloading is critical

15

for determining the turnover of the mature polyketide in the PKS machinery.8–12

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Substantial effort has been undertaken to elucidate the biosynthetic machinery of

17

PKS owing to the discovery of new and improved polyketide analogs with protein

18

engineering.13–16 By the turn of the century, a large number of polyketide analogs had

19

been obtained by substituting individual enzymatic domains or entire modules with

20

different building block specificity, or by deleting various enzymatic functions.17–20 On

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the other hand, exploration in optimizing PKS substrates through substrate engineering

22

is underway to minimize hydrolysis and other undesired side reactions.21–23 Notably,

23

PKS TEs have received considerable attention in the recent past, since TE-catalyzed

24

macrocyclization is essential for successful biosynthesis of new macrocyclic

25

compounds. There have been substantial in vivo and in vitro data of PKS TEs. Most

26

TEs exhibit the flexibility of substrate loading, but have the weakness of product

27

selectivity. More importantly, hydrolysis process seems rather facilitated compared to

28

macrocyclization in

29

N-acetylcysteamine thioester (SNAC) substrate.12,24–26 In 2003, Boddy and his

a large number of in vitro experiments using the

4


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co-workers first found that epothilone TE could catalyze macrolactonization in vitro.21

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Aldrich et al. further confirmed pikromycin thioesterase has the ability to

3

macrocyclizate SNAC-seco-10-deoxymethynolide exclusively.27 In 2006, Substrates

4

SNAC-1 and SNAC-2 were observed to proceed exclusively through macrocyclization

5

and hydrolysis, respectively.27,28 Interestingly, the unique difference in them lies in the

6

group connected to C7, where the former is C7-carbonyl group and the latter

7

C7-hydroxyl group (Figure 1). Also other experiments have reported that

8

macrocyclization of thioesterase is so specific that a little change on the substrate can

9

result in completely different final product.22,29

10

11 12

Figure 1. Macrocyclization and hydrolysis catalyzed by DEBS TE with substrates 1

13

and 2. Native substrate of DEBS TE is designated as 3. The exclusive products are

14

shown in dashed line.

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A two-step mechanism has been proposed for the TE-mediated formation of

17

macrocyclic polyketides.8,30,31 The first step is acylation of a linear polyketide thioester

18

at a universally conserved serine residue in the catalytic triad of the TE to generate an

19

acyl-enzyme intermediate, which can be intensely stabilized for a considerable time.32 5


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The second step is a nucleophilic attack of an intramolecular hydroxyl group leading to

2

cyclization, or hydrolysis of the acyl-enzyme intermediate when no efficient

3

intramolecular nucleophile is available. Investigations have attempted to uncover the

4

characteristics of the substrate selectivity for macrocyclization of 12 or 14-membered

5

macrocyclic narbonolide by DEBS TE. Unlike the NRPS TEs, where substrate

6

pre-organization appears to drive macrocyclization, the DEBS TE requires specific

7

enzyme substrate interactions for macrocyclization. According to their docking results,

8

Tsai et al. suggested that polyketide chain could be anchored by hydrogen bonds

9

network and subsequently catalyzed by an internal nucleophilic attack,30,33 while Wang

10

et al. demonstrated that the substrate interacts primarily through hydrophobic packing

11

with the enzyme, rather than hydrogen-bonding interactions.34

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Although biochemical studies have examined the function of PKSs, the molecular

13

basis of the mechanism underlying TE catalysis remains unclear. It should be noted that

14

in recent years few theoretical studies have explored the catalytic mechanism of

15

thioesterase, especially for macrocyclization.35,36 We wonder, for example, in the

16

context of TE, what determines the macrocyclization or hydrolysis of substrate and its

17

specificity? What is the catalytic mechanism and how large is the energy barrier?

18

Insights into these questions will lead to better understanding of substrate specificity,

19

TE mechanism and the biosynthetic machinery of PKS.

20

To understand the releasing process catalyzed by PKS TEs, we combined

21

molecular dynamic (MD) simulations with quantum mechanics (QM) and QM/MM

22

calculations on systems DEBS-TE-1, DEBS-TE-2 (including chiral substrates (R)-2

23

and (S)-2) and DEBS-TE-3 (native substrate of DEBS TE) in an aqueous environment.

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Herein, we report the conformational transitions of both DEBS-TE and its substrates by

25

induced-fit interactions for mutual recognition and catalytic preparation. QM

26

calculations were employed to investigate the detailed catalytic mechanism. Transition

27

states and key intermediates, especially a pre-reaction state, were located in calculated

28

potential energy surface. The possible molecular mechanism of macrocyclization

29

process was finally proposed. These results are in good agreement with biochemical 6


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and structural studies. Our study elucidates the catalytic mechanism of DEBS-TE with

2

its substrates, provides full insight into the macrocyclization process of DEBS-TE, and

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increases comprehensive understanding of type I PKS TEs in engineered PKS pathway.

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MATERIALS AND METHODS

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System preparation: Initial protein structure of DEBS TE was taken from the X-ray crystal data

7

(PDB entries: 1KEZ).33 The substrates of 1, 2 and 3 covalently bind with residue Ser142, which is

8

terminal blocked by adding N-terminal cap (-CO-CH3) and C-terminal cap (-NH-CH3) (Supporting

9

Information, Figure S1). A local conformational search strategy was used for the

10

covalently-connected macromolecular system with the following steps: 1) the classical

11

conformational search was conducted for the isolated substrates with a systematic search method

12

(CAESAR)37 encoded in Discovery Studio 3.5 and obtained 3000 ligand conformations; 2) then the

13

top 20 low-energy conformations were further optimized with PM3 method38–40 in Gaussian 0941;

14

and 3) the lowest-energy conformations were placed into the protein, to construct the initial

15

structure with adding covalent connection between Ser142 and ligand. Four complex systems were

16

constructed, DEBS-TE-1, DEBS-TE-(R)-2, DEBS-TE-(S)-2 and DEBS-TE-3, which were slightly

17

adjusted to avoid severe steric repulsion by a small single bond rotation (C2-O3) of less than 5o

18

when the substrate and enzyme connected together (Figure S2). Files were prepared in the tleap

19

module of the AMBER program suite.42

20

Parameter preparation for substrates 1, 2 and 3 was performed by using the Antechamber

21

package.43,44 First, the optimization was performed by the Gaussian 09 program at the level of

22

HF/6-31G(d)45-47 and then the electrostatic surface potential (ESP) charge was calculated. A

23

two-step restrained electrostatic potential (RESP)48 charge fitting was applied to the substrates.

24

Finally, the missing bond and dihedral parameters were generated by using the Antechamber

25

package (Table S1, S2, S3 and S4).

26

MD simulation and trajectory analysis: MD simulations using the AMBER program suite with

27

ff03.r1 force field were carried out to the prepared structures of DEBS-TE-1, DEBS-TE-2 and

28

DEBS-TE-3. All ionizable side chains were maintained in their standard protonation states at pH

29

7.0. The proteins were solvated in a cubic box of TIP3P water molecules with a water thickness 7


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extending at least 10 Å away from the protein surface. Sodium ions were then added to the system

2

as counterions to create a neutral simulation system.

3

To avoid any instability that might occur during the MD simulations, the solvated system was

4

subjected to 10000 steps minimization and changed from the steepest descent algorithm to

5

conjugate gradient after 1000 cycles. Then, the system was gradually heated from 0 K to 300 K in

6

50 ps controlled by Langevin dynamics with a collision frequency of 2 ps-1. Next, the system was

7

switched to constant pressure and temperature (NPT) and equilibrated for 50 ps to adjust the

8

system to the correct density. Finally, the production simulations were carried out in the absence

9

of any restraint under NPT conditions and 100 ns MD simulation was conducted. Each simulation

10

was repeated 4 times and one of them was extended to 500 ns simulation. The Particle Mesh

11

Ewald (PME) method49 was employed to calculate long-range electrostatic interactions, and the

12

lengths of bonds involving hydrogen atoms were fixed with the SHAKE algorithm50. During

13

simulations, an integration time step of 2 fs was adopted, and structural snapshots were flushed

14

every 500 steps (1 ps). The non-bonded cutoff was set to 10.0 Å. This protocol was applied to all

15

of the simulation systems and all MD simulations were performed using the parallel version of

16

PMEMD.cuda in the AMBER12 suit.

17

The hydrogen bonding analysis of the simulation trajectory was carried out with the cpptraj

18

in Ambertools1451 and the default options (the donor-acceptor distance is < 3.0 Å and

19

acceptor-donor-hydrogen angle is > 135º). A mass-center definition of a hydrophobic interaction

20

was used in this paper: two hydrophobic groups are considered to be interactive if the mass-center

21

distance ≤ 6.5 Å. Root mean-square deviation (RMSD)-based clustering was performed by the

22

widely accepted average linkage clustering algorithms. The representative structures were further

23

extracted to describe the conformational change of substrate.

24

Conformational stability calculation: A scheme of molecular mechanics Generalized-Born

25

surface area (MM-GBSA)52,53 calculation was conducted on the substrate-enzyme complexes to

26

estimate the conformational stability. The MMGBSA.py method implemented in the AMBER12

27

program was applied to compute the total of free energies (Gtot) in the solution and decomposed to

28

substrates 1, 2 residues. The relative energy was then computed on each cluster of conformations

29

with the reference to the beginning structure, using the resulting total energy with the MM-GBSA

30

method. 8


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QM and QM/MM calculation: The original model of the QM calculation was extracted as

2

representative structures from the dominant clusters obtained from MD simulations. Because a

3

molecule of water was present in the MD simulation and remained in the hydrophilic part of

4

catalytic cavity, the water molecule was retained in the QM model to investigate its role in the

5

reaction. It was found that the water molecule also stabilizes the system through

6

hydrogen-bonding interactions with substrates. All the QM calculations were performed with

7

B3LYP54-56/6-31G(d) method.

8

QM/MM calculations were performed using a two-layered ONIOM method57-61 encoded in

9

the Gaussian09 program. The representative structures of dominate clusters were selected based

10

on Cα and further optimized at the ONIOM (B3LYP/6-31G(d):Amber) level. The QM region

11

included the substrates 1 or 2 and the anionic carboxymethyl group (–CH2–COO-) of Asp159, the

12

side chain of His259 and a water molecule around the His259 (Figure S3). The QM system of

13

substrate 1 has a total of 74 atoms and substrate 2 has 76 atoms. This brought the total charge of

14

the QM system to -1 and the charge of total system is -17, since Na+ ions were abstracted from the

15

structure.

16

The QM region was calculated at the level of density functional theory with the B3LYP

17

exchange-correlation functional and 6-31G(d) basis set. The remainder of the system (MM region)

18

was treated with the AMBER Parm99 force field. A total of 7991 atoms for DEBS-TE-1 and 7993

19

atoms for DEBS-TE-2 were included in the QM/MM calculations. The electrostatic interactions

20

between the QM and MM regions were calculated by the electronic embedding method, which

21

treats the polarization of the QM region by the MM region with scaled partial atomic charges of

22

MM atoms. The response of the QM region is treated with the Merz-Singh-Kollman scheme for

23

charge fitting to produce the changing partial charges of the QM atoms. Thus, the B3LYP

24

calculations of the core layer would provide a reasonable energy potential for bond formation and

25

breakage, and the AMBER level for the outer layer could avoid severe steric effect and consider

26

hydrophobic interaction from remaining residues of DEBS TE.

27 28 29

RESULTS AND DISCUSSION To investigate the macrocyclization process of substrates 1 and 2 catalyzed by 9


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1

DEBS TE, two complex systems (DEBS-TE-1 and DEBS-TE-2) were conducted for

2

500-ns MD simulations and their results were compared to DEBS TE with its native

3

substrate 3 (DEBS-TE-3). QM and QM/MM calculations were used to study the

4

molecular mechanism of releasing step in macrocyclization. A potential energy

5

surface was calculated to confirm pivotal pre-reaction state. Finally, based on our

6

computational results, a detailed mechanism of DEBS TE catalyzed macrocyclization

7

was proposed.

8

Conformational alternations of DEBS TE in binding with substrates

9

According to the root-mean-square deviation (RMSD) values, the systems of

10

DEBS-TE-1, DEBS-TE-2 and DEBS-TE-3 have all reached their respective

11

equilibriums after 10 ns simulations (Figure S4). To ensure the accuracy of our results,

12

the first 20 ns simulation is not considered in our analysis. Our MD simulations of

13

DEBS-TE-1 and DEBS-TE-2 displayed the alternations of the substrate channels,

14

especially in the lid region, which is composed of two helices (helix 6 and helix 7) and

15

a loop, designated as L1, L2 and lid loop respectively (Figure 2A), observed in both

16

classical and non-classical TEs.62 The root-mean-square fluctuation (RMSF) values

17

indicated that (1) the lid loop adopts relatively larger fluctuations compared with L1

18

and L2 (Figure 2B), suggesting the lid loop might be a key factor in substrate

19

recognition; (2) The RMSF values of L1 ranging from residues 183 to 190 are much

20

larger than those for the remaining L1 part and L2 domain, indicating larger spatial

21

availability at their neighborhoods in the cases of substrates toward macrocyclization,

22

especially at a closed state (Figure 2D). Moreover, the exactly different conformational

23

evolutions of DEBS-TE-1 and DEBS-TE-2 were observed in the first 100 ns

24

simulation. The channel size of substrate 1 was found to gradually increase after ~20 ns

25

equilibrium, till the channel reached an “open” state, while in the case of DEBS-TE-2,

26

the size of substrate channel continuously reduced in the opposite direction and finally

27

resulted in a “closed” state. Interestingly, the channel of DEBS-TE-3 shows an “open”

28

state from beginning to ~150 ns (Figure 2E). These results indicated that the lid region

29

may play an important role in recognition of the substrate through induced fit 10


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interaction and the distinct conformational changes between open and closed states

2

may further contribute to macrocyclization or hydrolysis pathway.63-65 It should be

3

noted that the open-closed motion doesn’t coincide with the formation and breakdown

4

of pre-reaction state in a longer 500 ns simulation, and it is protein dynamic motion that

5

causes the open-closed flip and formation of the pre-reaction state. In the following

6

discussion, we focus on how pre-reaction state appears in the MD simulation,

7

especially, in the first 100 ns trajectories.

8 9

Figure 2. (A) Structure of DEBS TE with active site triad (Ser142-His259-Asp169) and

10

lid region. (B) The RMSF values of DEBS-TE-1, DEBS-TE-2 and DEBS-TE-3 in

11

500-ns MD simulations, where lid region are shown in red (L1), blue (lid loop) and

12

orange (L2). (C) Conformational alternations of DEBS TE in DEBS-TE-1,

13

DEBS-TE-2 and DEBS-TE-3. (D) The RMSF values of lid region in three states (open,

14

middle, and closed). (E) The statistical percentage of three states in every 10 ns of

15

500-ns simulation. The line chart under the bar in the DEBS-TE-1 and DEBS-TE-3

16

shows the population of the hydrogen bonding between C11 hydroxyl and His259

17

(rO-N < 3.5 Å). 11


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1 2

Interactions between DEBS TE and the substrates

3 4

Figure 3. Open and closed conformations of DEBS-TE-1 and DEBS-TE-2.

5 6

Effort has been focused on the molecular basis of the interactions between lid

7

region and substrate. By comparing the residue trajectories of DEBS-TE-1 between

8

the open and closed states (Figure 3), Arg193 of the lid loop was found to gradually

9

slip into the active site and expand the entrance of the substrate channel by disruption

10

of the hydrophobic interaction between Phe260 and Ile79. On the other hand, a

11

hydrophobic pocket composed of the residues Leu183, Leu186 and Leu190, which

12

distributed at the head, middle and tail of L1 region, were detected to function as a

13

pincer to capturing and stabilizing the substrate 1 in the open state. To sum up,

14

substrate 1 and these key residues of the lid region undergoes a significant

15

conformational transition, bringing the enzyme into a functional open state and finally 12


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ACS Catalysis

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triggering the macrocyclization process of DEBS-TE-1.

2

However, this is not the case for DEBS-TE-2 (Figure 3), where Arg193 was

3

observed to escape away from the active site of DEBS TE. In addition, the phenyl of

4

Phe260 was found to flip approximately 90 degrees to cover the entrance of the

5

substrate channel by interaction with Ile79. This hydrophobic interaction played a key

6

role in controlling the switch of the substrate channel. Additionally, another

7

hydrophobic interaction between Gln264 and Thr195 was found in a closed state of

8

DEBS-TE-(R)-2.

9 10

Figure 4. Hydrogen bonding interactions and hydrophobic interactions in closed (1I)

11

and

12

H2=Tyr171@O:1@H28;

13

（Plotting by using ligplot+66）

open

(1IV)

state

of

DEBS-TE-1,

where

H3=His259@Nε2:1@H33;

H1=Leu183@O:1@H33; H4=1@O1:Arg193@HH22.

14 15

The interactions between DEBS TE and the substrates were further investigated.

16

For substrate 1 (Figure 4), two primary hydrogen bonding interactions were found in a 13


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1

closed state among the hydrogen atoms of substrate 1. These atoms were interacting

2

with oxygen atoms of Leu183 (H1, 84.3 %) and Tyr171 (H2, 69.7 %), and in open

3

state, they were gradually replaced by hydrogen bonding interaction between C11

4

hydroxyl group and His259 (H3, 76.7 %), as well as Arg193 (H4, 55.0 %). We

5

propose that the hydrogen bonding H3 helps to promote the conserved

6

His259-Asp169 dyad to deprotonate the C11 hydroxyl group, increase substrate 1

7

nucleophilicity, and facilitate the macrocyclization. It should be noted that the

8

formation of hydrogen bonding between the terminal hydroxyl group and His259

9

accompanies with dynamic motion of open-closed states (Figure 2E). On the other

10

hand, five major hydrophobic interactions were found in closed state, indicating the

11

critical role of hydrophobic interaction in recognition and stabilization of the substrate.

12

Interestingly, almost all of these interactions remain in the open state, except that for

13

Phe260. After the conformational transition from the closed to open state, especially

14

the rotation of the C10 methyl and the C11 ethyl, substrate 1 was found to settle down

15

in a hydrophobic pocket which is formed by Leu183, Leu186, and Leu190. Therefore,

16

we proposed that hydrophobic interactions probably play a critical role in the

17

conformational transition and mutual recognition between DEBS TE and the

18

substrates. Besides, the hydrogen bonding and hydrophobic interactions in

19

DEBS-TE-(R)-2 are shown in Figure S5. In brief, the hydrophobic pocket (Ala77,

20

Leu183, Leu186 and Leu190) and gate-controller Phe260 acts together as five fingers

21

of a “hand” to pushing the opposite hydroxyl group to the hydrophilic reactive

22

chamber formed by the catalytic triad. This is in agreement with suggestions of Wang

23

et al., which demonstrated that the substrate interacts primarily through hydrophobic

24

packing with the enzyme, rather than hydrogen-bonding interactions.30,34 Furthermore,

25

these results also highlight the importance of these hydrophobic amino acids.

26 27 28 29 30 14


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ACS Catalysis

1

Conformational transitions of substrates

2 3

Figure 5. (A) Representative conformations of DEBS-TE-1 and DEBS-TE-3. (B)

4

Representative conformations of DEBS-TE-2. (C) The distances rO-N (terminal

5

hydroxyl O to His259 Nε) and rO-C (terminal hydroxyl O to carbonyl C1) in

6

DEBS-TE-1 (black) and DEBS-TE-3 (green).

7 8

Next, we focused on the conformational transitions of substrates, the clustering

9

algorithm was utilized to collect all typical substrate conformations mainly based on a

10

100 ns MD simulation. Five representative conformers (1I, 1II, 1III 1IV and 1IV') for

11

DEBS-TE-1 system were obtained (1IV' was found in the 500 ns MD simulation). The

12

major difference of the first four conformations lies in the rotation of substrate tail,

13

especially the orientation of C10 methyl and C11 ethyl (Figure S6). To be precise, in

14

substrate conformers from 1I to 1IV, the C10 methyl rotated upside down which was

15

accompanied by the swinging of C11 ethyl. In their proper orientations, a hydrogen

16

bonding emerged between the active C11 hydroxyl group and Nε of His259 in catalytic 15


ACS Catalysis

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1

triad, which is designated as H3 above. The distance rO-N between O and Nε decreased

2

from ~6.0 to ~2.5 Å during the transition process, indicating the formation of the

3

hydrogen bonding, which remains stable in the following ~100 ns MD simulation.

4

After ~270 ns MD simulation, another representative conformer 1IV' (the pre-reaction

5

state) was found possessing the same hydrogen bonding between C11 hydroxyl group

6

and Nε of His259 (Figure 5A), and the distance rO-C between O of C11 hydroxyl group

7

and C1 decreases from ~6.5 to ~5.0 Å, suggesting the superiority of nucleophilic

8

attack. Furthermore, in the DEBS-TE-3 system this similar structure was also observed

9

from the beginning of the simulation to the end with even shorter nucleophilic distance

10

of rO-C (~3.0 Å) compared with 1IV'. Also, two groups of substrate conformers 2I, 2II,

11

2III and 2IV were obtained (Figure S7). Structures of (R)-2IV and (S)-2IV are shown in

12

Figure 5B. Similarly, the rotation of substrate C10 methyl and C11 ethyl were

13

observed, but no hydrogen bonding between C11 hydroxyl group and Nε of His259 was

14

detected. Additionally, in one simulation of (S)-2 we observed an escape transition of

15

substrate from entrance to exit, which strongly suggests preference for hydrolysis

16

(Figure S8).

17 18 19

Table 1. Relative energetics calculated in the MM-GBSA scheme (referred to Conformer I, in kcal/mol). Conformer

I

II

III

IV

1 (R)-2 (S)-2

0.00 0.00 0.00

2.39±0.17 4.18±0.20 0.95±0.16

2.61±0.18 3.77±0.20 0.41±0.16

-0.49±0.18 3.45±0.21 1.11±0.16

20 21

We further evaluated relative stability of clustered conformations of DEBS-TE-1

22

with a MM-GBSA scheme (Table 1). The climbing energy from conformer 1I to

23

conformer 1III suggests the unfavorable interaction between substrate 1 and DEBS TE

24

in the formation of 1IV conformer. This barrier facilitates the rotation of C10 methyl

25

and C11 ethyl, through a significantly conformational transition, and further into a

26

functional open state. In the case of DEBS-TE-2, the relative energy fluctuates 16


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ACS Catalysis

1

dependently on the stereochemistry but both not to an energy sink, indicating an

2

incidental conformational alteration among them. Considering the conformational

3

change of lid region, it is proposed that the energy barrier could be overcome by

4

combining the effort of the hydrogen bonding and hydrophobic interaction between

5

substrate 1 and DEBS-TE, which was shown by the low-energy conformer (1IV).

6 7

Structure analysis of substrate 1

8

Next, we carefully examined the substrate conformers 1IV and 1IV', where a

9

hydrogen bonding between the active C11 hydroxyl group and Nε of His259 was

10

discovered to stabilize the conformations. Not so with 1IV, whose nucleophilic attack

11

distance between hydroxyl group of C11 and C1 of substrate 1 is larger than 6 Å, the

12

hydroxyl group of 1IV' could rotate to a proper position fit for the nucleophilic attack

13

with the distance less than 5.0 Å. Therefore, we constructed a computational model to

14

investigate the conformational transition between conformers 1IV and 1IV'. The model

15

was extracted from conformers 1IV, designated as R, including a water molecule, the

16

terminal groups of Asp159 and His259, as well as substrate 1, which was covalently

17

linked to Ser142.

18

A two-dimensional potential energy surface was calculated to aid in

19

understanding the rotation of substrate 1, especially C10 methyl, C11 ethyl, and C11

20

hydroxyl. The potential energy surface was calculated by defining the dihedral angels

21

d1 (1C-2C-3C-4C) and d2 (2C-3C-5C-6O) as the reaction coordinates (Figure 6), which

22

represent the rotation of C10 methyl and C11 hydroxyl, respectively. The potential

23

energy surface with the key structures along the reaction pathway, including R, TS,

24

IM1 and IM2, are shown in Figure 6. The dihedral angel of d1 was found to be 112.3º in

25

the optimized structure R and 72.2º in the optimized intermediate IM1, and the dihedral

26

angel of d2 is -178.9º in R and -53.1º in IM1. The transition state TS is located at d1 =

27

98.2º and d2 = -101.1º. The calculated potential energy barrier of rotation of substrate

28

tail was ∆G≠ = 11.6 kcal/mol, suggesting that the conformational transition could

29

proceed spontaneously. Our computational results are in strong agreement with the MD 17


ACS Catalysis

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1

simulations.

2

3 4

Figure 6. Two-dimensional potential energy surface defining the dihedral angels d1

5

(1C-2C-3C-4C) and d2 (2C-3C-5C-6O) as the reaction coordinates and the key

6

structures of R, TS, IM1 and IM2 along the reaction path. Black dashed lines represent

7

the nucleophilic attack distance between O atom of C11 hydroxyl group and C1 of

8

substrate 1.

9 10

Interestingly, another optimized structure of IM2 is located in the potential energy

11

surface, which is 1.2 kcal/mol lower than IM1 in energy and very close to the structure

12

of 1IV'. This structural reorganization made the hydroxyl group of C11 is more

13

propitious to attack the C1 atom of substrate 1. It is noteworthy that the nucleophilic

14

attack distance decreased from 6.76 Å in IM1 to 5.80 Å in IM2, which will help the

15

formation of a tetrahedral intermediate, and facilitate the cyclization process. 18


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ACS Catalysis

1 2

Pre-reaction state

3

The conformational evolution of substrate conformers 1IV to 1IV' (the pre-reaction

4

state) have been uncovered in one of our MD simulations (Figure S9) and the

5

additional 400 ns simulations. According to the QM computational results, this

6

conformational transition from 1IV to 1IV' (equivalent to R and IM2) can proceed

7

spontaneously through rotation of substrate tail with calculated potential energy barrier

8

of 11.6 kcal/mol. To sum up, it should be proposed that conformer 1IV is favorable in

9

energy and conformer 1IV' is favorable to cyclization process. Different from 1IV, the

10

C10 methyl of 1IV' rotated upside accompanied by the swing of C11 ethyl, meanwhile

11

the hydrogen bonding between C11 hydroxyl group and Nε of His259 remained at

12

rest. Therefore, it is proposed that formation of the conformer 1IV' is not only critical

13

to the precise recognition of enzyme-substrate through conformational transitions, but

14

also significant to the cyclization process, since substrate 1 was observed to

15

exclusively obtain macrocyclic polyketides. Also it should be noted that the 1IV'

16

conformation assembles in a similar manner to the substrate pre-organization in some

17

non-ribosomal peptide synthases (NRPS) TEs, where a key intramolecular hydrogen

18

bonding interaction has been discovered to facilitate macrocyclization in favoring of

19

hydrolysis.

20

Additionally, QM/MM calculations were carried out to verify the structures and

21

energies of 1IV'. After the conformational transition, the nucleophilic attack distance

22

decreased from 7.01 to 4.85 Å in DEBS-TE-1. Besides the structural similarity, the

23

energy gap between conformers 1IV to 1IV' was calculated to be 5.0 kcal/mol, which is

24

close to 4.1 kcal/mol between R and IM2 (Figure 7). These results are in good

25

agreement with each other and indicate the reliability of our computational models.

26

Furthermore, the artificial structures of 2IV and 2IV' were optimized by using the same

27

methods. The calculations indicated that both (R)-2IV' and (S)-2IV' could be stable at the

28

pre-reaction state, which are even more favorable in measures of energy when

29

comparing to 2IV and 2IV'. 19


ACS Catalysis

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1

2 3

Figure 7. Optimized structures of DEBS-TE-1 and DEBS-TE-2 by QM/MM method.

4 5

Altogether, our computational results demonstrated that the unique difference

6

between C7-carbonyl group of substrate 1 and C7-hydroxyl group of substrate 2 was

7

responsible for the diverse mutual recognitions between enzyme and substrates, which

8

ultimately contributed substrate 1 to move to a pre-reaction state and substrate 2 not to

9

do so. One possible reason is that the conjugation between C7 carbonyl group and

10

C8=C9 double bond promotes the conformational partition of pre-reaction state of

11

substrate 1. Furthermore, the results obtained from 100-ns simulations of GXG without

12

protein (where G is glycine and X is substrates 1 or 2) with three repetitions indicated

13

that the populations of pre-reaction-like structures in substrate 1 (the distance of C11 20


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ACS Catalysis

1

hydroxyl to C1 of substrate < 4 Å) were much higher than that in 2 (Table S5),

2

suggesting that C7 carbonyl group of substrate 1 has a more tendency to rotate the tail

3

close to C1 than C7-hydroxyl group of substrate 2. And we think the hydrophobic

4

protein environment in TEs will increase the selectivity of C7 carbonyl group to

5

pre-reaction states. In MD simulations of DEBS-TE-2, neither the 2IV nor 2IV'

6

conformations could be obtained, indicating the formation of the pre-reaction state

7

might play the key role in macrocyclization catalyzed by DEBS TE.

8

It should be noted that the pre-reaction states appear in the both MD trajectories

9

of DEBS-TE-1 and DEBS-TE-3. Leu183 and Val170 interact with the C2 methyl of

10

substrates 1 and 3, leveraging the substrate arms towards His259; Leu190 in

11

DEBS-TE-1 and Phe191 in DEBS-TE-3 provide hydrophobic effect pushing the

12

hydroxyl tail away and keeping water out of catalytic triad. Moreover, subtle

13

differences were observed about Ala143 in the formation of pre-reaction state. As

14

shown in Figure 10S, Ala143 and Ala77 face C7 carbonyl of substrate 1, while only

15

Ala77 pushes C9 carbonyl of substrate 3. Alternatively, in the case of 3, Ala143

16

provides amide N-H to form hydrogen bond with oxygen of C1 carbonyl (the same

17

hydrogen bonding to stabilize oxyanion of a charged tetrahedral intermediate) and

18

exposes the C1 atom for nucleophile. Oppositely, Ala143 restricts mobility of C7

19

carbonyl of substrate 1 and thereby decreases accessibility of C11 hydroxyl to C1.

20 21

Macrocyclization

22

Our computational model was also used to study the molecular mechanism of

23

macrocyclization with DFT calculations. Key structures along the reaction pathway

24

including two transition states (1IV'-TS1, 1IV'-TS2) and a charged tetrahedral

25

intermediate (1IV'-IM) were fully optimized and structures of 1IV-TS1, (R)-2IV'-TS1

26

and (S)-2IV'-TS1 are presented to compare with the pre-reaction state (Figure 8).

27

Compared with 1IV-TS1 and 1IV'-TS1, a prominent repulsion is found between C3

28

methyl and C11 ethyl in 1IV-TS1, while the repulsion is considerably milder in

29

1IV'-TS1, since the position of C11 ethyl is replaced by C10 methyl (Figure S11). To 21


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

1

be precise, the distance between C1 and O of C11 hydroxyl group is 2.06 Å in

2

1IV'-TS1, much larger than that of 1IV-TS1 (1.87 Å), suggesting that the pre-reaction

3

state 1IV' with the proper orientation of C10 methyl and C11 ethyl will decrease the

4

steric repulsion and help to stabilize the transition state. As expected, 1IV'-TS1 is

5

found to be much lower in free energy than 1IV-TS1 by 11.6 kcal/mol, when the

6

solvent effects are considered. Furthermore, the calculations of (R)-2IV' and (S)-2IV'

7

indicated that the chirality doesn’t result in a significant difference in free energy, and

8

the free energy of the macrocyclization via (R)-2IV'-TS1 and (S)-2IV'-TS1 is calculated

9

to be 10.9 and 9.1 kcal/mol, respectively, which is close to that via 1IV'-TS1 9.9

10

kcal/mol. These results strongly demonstrated that the pre-reaction state is critical to

11

macrocyclization, and whether substrates 1 and 2 could develop to the pre-reaction

12

state, seemed to decide whether macrocyclization or hydrolysis occurred.

13

14 15

Figure 8. Optimized key structures in DEBS-TE-1 (A) and DEBS-TE-2 (B).

16 17

In addition, the intermediates were found to be slightly lower in energy than the

18

transition states, suggesting the reversible reaction (i.e. the dissociation of the

19

intermediate state) could easily occur.32,67 Also the transition state of the product 22


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ACS Catalysis

1

release was obtained and designated as 1IV'-TS2, the energy barrier was calculated to

2

be 0.3 kcal/mol. On the other hand, the final macrocyclization product were optimized

3

and designated as 1IV'-P. It can be seen that 1IV'-P was 17.6 kcal/mol lower in free

4

energy than 1IV'-R, and 2.6 kcal/mol lower than 1IV-P, which is in notable agreement

5

with the native macrocyclization product 10-Deoxymethynolide catalyzed DEBS TE.

6

These results demonstrated that the final macrocyclization product, which is

7

preponderant in energy, should be obtained through pre-reaction state.

8 9

Catalytic Mechanism

10

Taking these data together, we proposed a detailed molecular mechanism of the

11

releasing process of macrocyclization catalyzed by DEBS TE: (1) the induced-fit

12

mutual recognition between the enzyme (DEBS TE) and substrate 1 was observed

13

through a critical hydrogen bonding interaction between C11 hydroxyl and Nε of

14

His259, and a hydrophobic pocket composed of residues Leu183, Leu186 and Leu190

15

in lid region, which was found to accommodate substrate 1 appropriately,

16

accompanying the substrate channel gradually reached to an “open” state; (2) next,

17

the conformational transition from 1IV to a pre-reaction state 1IV' is proposed to be the

18

key step with energy barrier of 11.6 kcal/mol. It would be possible to overcome by

19

combining the effort of hydrogen bonding and hydrophobic interaction between 1 and

20

DEBS-TE; (3) accompanied with the deprotonation of pre-reaction state 1IV', the

21

nucleophilic attack proceeds and a charged tetrahedral intermediate forms, whose

22

energy barrier was calculated to be of 9.9 kcal/mol; and (4) finally, following the

23

decomposition of the transient intermediate, the final macrocyclic product is released

24

with a very low barrier of 0.3 kcal/mol. It is expected that mutation of Leu183,

25

Leu186 and Val170 (as to alanine) would change the yield ratio of macrocyclization

26

and hydrolysis in experiment, and mutation of Phe260 and Ile79 to hydrophilic

27

residue would damage the formation of pre-reaction state.

28 29

CONCLUSIONS 23


ACS Catalysis

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1

The final step in polyketide synthase (PKS)-mediated biosynthesis of

2

macrocyclic polyketides is TE-catalyzed macrocyclization of a linear polyketide acyl

3

chain. Although substantial effort has been undertaken to elucidate the structural

4

characteristics and substrate specificities of TEs, the molecular mechanism remains

5

unclear. To understand the macrocyclization process, we combined MD simulations

6

with QM and QM/MM calculations on complexes of DEBS TE and its substrates (1

7

and 2). Their results were compared to DEBS TE with its native substrate 3

8

(DEBS-TE-3). The induced-fit mutual recognition between enzyme and substrates

9

was uncovered through conformational transitions; a critical hydrogen bonding

10

interaction and a hydrophobic pocket in lid region were found to play important roles

11

in recognition of substrates. A potential energy surface was calculated to obtain

12

pivotal transition state and pre-reaction state (1IV') by DFT method. The

13

conformational transition from 1IV to 1IV' is proposed to be the critical step with

14

energy barrier of 11.6 kcal/mol. Accompanied with the deprotonation of the

15

pre-reaction state, nucleophilic attack proceeds, and the charged tetrahedral

16

intermediates forms. The energy barrier was calculated to be 9.9 kcal/mol. Then, the

17

intermediate decomposes with a very low barrier of 0.3 kcal/mol and the final

18

macrocyclic product releases. Finally, we proposed the possible molecular

19

mechanisms of macrocyclization. The computational results demonstrated that the

20

unique difference between C7-carbonyl group of 1 and C7-hydroxyl group of 2 were

21

responsible for the pre-reaction state through diverse mutual recognition between

22

enzyme and substrates, which ultimately led to a macrocyclic product (substrate 1)

23

and a hydrolysis product (substrate 2). It is proposed that macrocyclization catalyzed

24

by DEBS TE might be highly selective for a pre-reaction state, and whether it could

25

develop to the pre-reaction state seemed to decide the resulting configuration of the

26

substrate. These results are in good agreement with biochemical and structural

27

studies.14,34 Our study demonstrates the mutual recognition between DEBS TE and

28

substrates, provides full insight into macrocyclization process, and increase

29

comprehensive understanding of type I PKS TEs in engineered PKS pathway.

30 24


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ACS Catalysis

1

ASSOCIATED CONTENT

2

Supporting Information

3

The structures of substrates 1, 2 and 3 (Figure S1). The RMSD of 500 ns simulations

4

for DEBS-TE-1, DEBS-TE-2 and DEBS-TE-3 (Figure S4). The conformational

5

changes from conformers 1I to 1IV (Figure S6). The conformational changes in

6

DEBS-TE-2 (Figure S7). The conformational changes in DEBS-TE-(S)-2 show the

7

substrate’s transition from entrance to the exit (Figure S8). The comparison of

8

pre-reaction states in DEBS-TE-1 and DEBS-TE-3 (Figure S10). The key structures

9

of DEBS-TE-1 and DEBS-TE-2 along the reaction pathways (Figure S11). The atom

10

types and charges of substrates 1, 2 and 3 (Table S1-S4). This material is available

11

free of charge via the Internet at http://pubs.acs.org.

12 13

AUTHOR INFORMATION

14

Corresponding Author

15

*Email: [email protected]. Tel/Fax: +86-21-34207190.

16

Author Contributions

17

# X.P.C and T.S. contributed equally to this work.

18

YLZ and TS conceived and designed the investigation. XPC, TS, XLW, JW and QC

19

performed the energetic calculations and analyses and TS, XPC, LB and YLZ wrote

20

up the paper.

21

Notes

22

The authors declare no competing financial interest.

23 24

ACKNOWLEDGMENTS

25

The authors thank the National Basic Research Program of China “973”

26

(2012CB721005, 2013CB966802) and the National High-tech R&D Program of

27

China “863” (2012AA020403), Shanghai Municipal Council of Science and 25


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

1

Technology (13YZ032), National Science Foundation of China (21303101, 21377085

2

and J1210047), the China Postdoctoral Science Foundation (Grants 2014T70413 and

3

2014M561463) and the SJTU-HPC computing facility award for financial supports

4

and computational hours. The authors acknowledge Dr. Timo Törmäkangas for his

5

helpful revision on language.

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

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(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)

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30

FIGURE LEGEND

31

Figure 1. Macrocyclization and hydrolysis catalyzed by DEBS TE with substrates 1

32

and 2. Native substrate of DEBS TE is designated as 3. The exclusive products are

33

shown in dashed line.

34

Figure 2. (A) Structure of DEBS TE with active site triad (Ser142-His259-Asp169) and

35

lid region. (B) The RMSF values of DEBS-TE-1, DEBS-TE-2 and DEBS-TE-3 in

36

500-ns MD simulations, where lid region are shown in red (L1), blue (lid loop) and

37

orange (L2). (C) Conformational alternations of DEBS TE in DEBS-TE-1,

38

DEBS-TE-2 and DEBS-TE-3. (D) The RMSF values of lid region in three states (open,

39

middle, and close). (E) The statistical percentage of three states in every 10 ns of 28


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1

500-ns simulation. The line chart under the bar in the DEBS-TE-1 and DEBS-TE-3

2

shows the population of the hydrogen bonding between C11 hydroxyl and His259

3

(rO-N < 3.5 Å).

4


5

Figure 4. Hydrogen bonding interactions and hydrophobic interactions in closed (1I)

6

and

7

H2=Tyr171@O:1@H28;

8

（Plotting by using ligplot+66）

9

Figure 5. (A) Representative conformations of DEBS-TE-1 and DEBS-TE-3. (B)

10

Representative conformations of DEBS-TE-2. (C) The distances rO-N (terminal

11

hydroxyl O to His259 Nε) and rO-C (terminal hydroxyl O to carbonyl C1) in

12

DEBS-TE-1 (black) and DEBS-TE-3 (green).

13

Figure 6. Two-dimensional potential energy surface defining the dihedral angels d1

14

(1C-2C-3C-4C) and d2 (2C-3C-5C-6O) as the reaction coordinates and the key

15

structures of R, TS, IM1 and IM2 along the reaction path. Black dashed lines represent

16

the nucleophilic attack distance between O atom of C11 hydroxyl group and C1 of

17

substrate 1.

18


19


open

(1IV)

state

of

DEBS-TE-1,

where

H3=His259@Nε2:1@H33;

H1=Leu183@O:1@H33; H4=1@O1:Arg193@HH22.

29


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

Figure 1. Macrocyclization and hydrolysis catalyzed by DEBS TE with substrates 1 and 2. Native substrate of DEBS TE is designated as 3. The exclusive products are shown in dashed line.


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ACS Catalysis

Figure 2. (A) Structure of DEBS TE with active site triad (Ser142-His259-Asp169) and lid region. (B) The RMSF values of DEBS-TE-1, DEBS-TE-2 and DEBS-TE-3 in 500-ns MD simulations, where lid region are shown in red (L1), blue (lid loop) and orange (L2). (C) Conformational alternations of DEBS TE in DEBS-TE1, DEBS-TE-2 and DEBS-TE-3. (D) The RMSF values of lid region in three states (open, middle, and closed). (E) The statistical percentage of three states in every 10 ns of 500-ns simulation. The line chart under the bar in the DEBS-TE-1 and DEBS-TE-3 shows the population of the hydrogen bonding between C11 hydroxyl and His259 (rO-N < 3.5 Å).


ACS Catalysis

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ACS Catalysis

Figure 4. Hydrogen bonding interactions and hydrophobic interactions in closed (1I) and open (1IV) state of DEBS-TE-1, where H1=Leu183@O:1@H33; H2=Tyr171@O:1@H28; H3=His259@Nε2:1@H33; H4=1@O1:Arg193@HH22.(Plotting by using ligplot+66)


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

Figure 5. (A) Representative conformations of DEBS-TE-1 and DEBS-TE-3. (B) Representative conformations of DEBS-TE-2. (C) The distances rO-N (terminal hydroxyl O to His259 Nε) and rO-C (terminal hydroxyl O to carbonyl C1) in DEBS-TE-1 (black) and DEBS-TE-3 (green).


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ACS Catalysis

Figure 6. Two-dimensional potential energy surface defining the dihedral angels d1 (1C-2C-3C-4C) and d2 (2C-3C-5C-6O) as the reaction coordinates and the key structures of R, TS, IM1 and IM2 along the reaction path. Black dashed lines represent the nucleophilic attack distance between O atom of C11 hydroxyl group and C1 of substrate 1.


ACS Catalysis

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ACS Catalysis



Theoretical Studies on the Mechanism of Thioesterase-Catalyzed

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