Computational Insights into the Catalytic Mechanism of Bacterial

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Computational Biochemistry

Computational Insights into the Catalytic Mechanism of Bacterial Carboxylic acid Reductase Ge Qu, Mingxing Fu, Lili Zhao, Beibei Liu, Pi Liu, Wenchao Fan, Jun-An Ma, and Zhoutong Sun J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00763 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Journal of Chemical Information and Modeling

Computational Insights into the Catalytic Mechanism of Bacterial Carboxylic acid Reductase Ge Qu†#, Mingxing Fu‡#, Lili Zhao‡, Beibei Liu†, Pi Liu†, Wenchao Fan†, Jun-An Ma§, and Zhoutong Sun†* †Tianjin

Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China ‡Institute of Advanced Synthesis, School of Chemistry and Molecular Engineering, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing 211816, China §Department of Chemistry, Tianjin Key Laboratory of Molecular Optoelectronic Sciences, and Tianjin Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China ABSTRACT: Multi-domain carboxylic acid reductases (CARs) can reduce a wide range of carboxylic acids to corresponding aldehydes in the presence of ATP and NADPH. Recent X-ray structures of the individual (di)domains of Segniliparus rugosus CAR (SrCAR) shed light on the catalysis mechanism and revealed domain dynamics during the different states of the reaction. However, the details of the catalytic mechanism of each steps operated by corresponding domains are still elusive. Toward this end, several models were constructed based on the crystal structures, and molecular dynamics (MD) simulations along with density functional theory (DFT) calculations were employed to exploit the conformational dynamics and the catalytic mechanism of SrCAR concealed to static crystallography. We investigated the roles of the key residues in the substrate binding pocket involved in the adenylation and thiolation reactions, and especially determined the roles played by a non-conserved Lys528 residue in the thiolation step, which was further verified by site-directed mutagenesis. The reduction mechanism of SrCAR, including the nature of the transition states of hydride and proton transfer, was also explored theoretically using the DFT method B3LYP. The information presented here is useful as a guide for the future rational design of CARs.

INTRODUCTION As a versatile enzyme, carboxylic acid reductases (CARs) are capable of reducing a wide range of carboxylic acids to the respective aldehydes using the cofactors ATP and NADPH in mild conditions, which constitutes a very challenging transformation via chemical methods.1-3 CARs have shown a relaxed substrate tolerance, featuring in combination with other enzymes efficient ways to synthesize fuels, fine chemicals, pharmaceuticals and other valuable commodities in “attractive green chemical” routes.3 Typically, CAR is comprised of an N-terminal adenylation (A) domain, a thiolation (T) domain, and a C-terminal reductase (R) domain. The A domain is composed of a large N-terminal core catalytic domain (Acore) and a small mobile C-terminal subdomain (Asub).4 Quite differently, the T domain is attached a phosphopantetheinyl (PPT) arm to a conserved serine in the posttranslational modifications.5 The overall catalytic route for the reaction in CARs was first proposed by Venkitasubramanian in 2007,5 and was well accepted by other studies.6-11 Initially, the carboxylic acid substrate undergoes adenylation at the expensive of ATP, releasing pyrophosphate (PPi) and forming an acyl-AMP complex. The complex thereupon endures a nucleophilic attack by the PPT thiol at the carbonyl carbon, resulting in the formation of the covalently bound acyl-thioester. Next, the acyl-thioester is repositioned to the R domain by swinging the PPT arm, and then it goes through reductive cleavage by NADPH to yield the aldehyde product. Finally, the thioester arm swings back to the A domain, thereby setting up the next catalytic cycle (Scheme 1).

O R

O

ATP OH

R

O

R'-SH AMP

R

S

R'

O

NADPH H

+

R

H

R'-SH

Scheme 1. General catalytic model of CAR reaction. Since both the adenylation and thiolation reactions occur in the A domain pocket, a large-scale structural reorientation within the Asub and T domains is arranged to guarantee the two steps undergoing in order, which was reported by a recent crystallographic study in bacterial CARs.4 Taking Segniliparus rugosus CAR (SrCAR) as an example, in the adenylation step (PDB code 5MSW), the T domain is located distantly from the A domain, causing a flexible loop (A10) in the Asub domain, that contains a universally conserved lysine residue (A10 Lys), to rotate into the pocket for enabling the catalysis of adenylation. The A10 Lys in ANL (the Acyl-CoA synthetases, the NRPS adenylation domains, and the Luciferase enzymes) superfamily is supposed to track the negative charge on the initial complex, stabilize the transition states, and finally to release the PPi before yielding the AMPsubstrate complex.12-19 The catalytic role played by the invariant Lys629 (A10 Lys in SrCAR) in adenylation was supported by previous biochemical studies in various ANL enzymes, including acyl-CoA synthetase,13-16 luciferase17 and NRPSs.18,19 After the adenylation state, both the Asub and T domains rotate into the thiolation conformation (PDB code 5MSS). The rotation results in Lys629 about 23 Å away from the catalytic pocket, and places a hinge region (A8) located in the Asub domain to the catalytic center, priming the enzyme for thioesterification (Figure 1).

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reduction mechanism was proposed, which includes the nature of the transition states in the hydride and proton transfer steps.

RESULTS AND DISCUSSION

Figure 1. The intrinsic flexibility of SrCAR, rotating from adenylation to thiolation state. The dashed arrows depict domain motion from 5MSW (grey) to 5MSS (white). The displacement distance of the A10 Lys is 23 Å. A10 and A8 are colored in magenta and cyan, respectively. Orange surface indicates the A domain catalytic pocket. In the reduction step, a short loop region nearby the catalytic pocket of the R domain guarantees the reduction terminated at the aldehyde rather than the alcohol product, by regulating the movement of a conserved Asp residue. Due to the two-electron reductive activity by CAR, the aldehyde product is formed on hydride transfer from NADPH and proton transfer from solvent. Nevertheless, the nature of the transition states concerning hydride and proton transfer during the reduction step is yet to be uncovered, and the proton transfer path also remains elusive (Scheme 2). In contrast, the reduction mechanism of many other NADPHdependent reductases, including NRPS,20,21 short-chain dehydrogenases,22-24 cinnamoyl-CoA reductases,25 fatty acyl-CoA reductases, HMG-CoA reductase,26,27 and nitrile reductases,28,29 have already been elucidated. In fact, the CAR R domain may adopt the SDR reduction mechanism (Scheme S1a), as it strongly resembles the reductase domain of SDR family on the structural level.30 On the other hand, the R domain may also follow the CoA reductase catalytic mechanism (Scheme S1b), with respect to the similar acyl-thioester substrates. O R

S

R'

O

NADP-H H

+

R

H

H S

O R'

R'-SH

R

H

Scheme 2. Reduction catalyzed by CAR. Proton and hydride are remarked in blue and red colors, respectively. While the biochemical functions and crystal structures of CARs have been documented,6-11 a detailed investigation of how enzyme dynamics regulates the catalytic character at the atomic and molecular level is still obscure. Theoretical studies, such as molecular dynamics (MD) simulations and quantum mechanical (QM) calculations, can elucidate the mechanistic details with regard to enzyme structural and dynamic properties that may not be possible via experimental means.22,31-34 To our knowledge, no such relatively theoretical studies have been performed on the bacterial CARs to date. In this study, MD simulations along with QM calculations were applied in SrCAR to shed light on the mechanistic details. We investigated the roles of the active site residues in the adenylation and thiolation reactions, and especially determined that the less conserved Lys residue, located in the A8 motif, plays central roles in thioesterification, which was further confirmed by mutational analysis. Reduction in the R domain was modeled by QM, using an abbreviated QM model of the active sites. A

Conservation analysis and allosteric regulation in the A-T didomain. The A domain of CAR is closely related to the ANL superfamily members in terms of substrate specificity and structural features.12 Despite the low sequence similarity of the CAR A domains and other ANL family members, nine well conserved motifs can be easily recognized (Figure S1) by sequence alignment of SrCAR A domain with other crystallized ANL family members (Table S1). Following the conservation analysis, a number of key residues (Table S2) in SrCAR A-T didomain were annotated based on the previous studies of CAR and ANL enzymes.4,10,12 This result suggests that the annotated residues may execute potential functions during the reaction. The A-T didomain of SrCAR has been shown to utilize two specific catalytic conformations corresponding to the successive reactions. In details, the A-T didomain rotates to the adenylatedforming conformation, trigged by binding of ATP and the substrate. Upon formation of the adenylated-substrate complex, the release of PPi occurs (Figure S2a). As a consequence, it enables the Asub domain rotates to a new orientation for catalyzing the thiolation reaction.35 Such domain alternation conformational change was observed in structural and functional studies of other ANL family members.12,18,36-39 The molecular surface of the catalytic pocket is predominantly covered with polar and positive charges (Figure S2b). Positive charge regions (blue cycle, Figure S2c) are complemented with the negatively charged path on the T domain surface (red cycle, Figure S2c). When the Asub domain rotates to form a second conformation corresponding to thiolation (PDB code 5MSS), X-ray crystal structures of SrCAR revealed a large-scale structural reorientation (Figure 2 and Movie S1). While protein dynamics can mediate long-range allostery, and dynamics cross-correlation map (DCCM) is a powerful means to investigate the covalent connectivity in domain–domain communication,40,41 we therefore computed DCCMs for adenylation and thiolation states, respectively (Figure S3). Compared to the Acore region, the Asub and T domains clearly showed positive correlations from adenylation to thiolation state, indicating the changes occurring at the binding pocket are transmitted toward the T domain to trigger the enzyme conformational changes. As a consequence of the domain rearrangement, the active sites (Table S2) in the catalytic pocket can be divided into two groups: flexible sites (red inset, Figure 2) and relatively stable sites (blue inset, Figure 2). Notably, the Asub domain was thought to be rotated ~165o at the A8 Lys528 hinge region,2 however, Lys524 and Leu529 were also showed dramatically changed conformations (more than 60 degrees) based on an analysis of the phi-psi backbone dihedral torsion angles (Table S3).


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Figure 2. The active sites in SrCAR A-T didomain. The adenylation (PDB code 5MSW) and thiolation (PDB code 5MSS) conformations are marked in green and blue colors, respectively. The red inset indicates the sites in a large movable manner during structure rotation. In contrast, the relatively stable residues are shown in the blue inset. Molecular dynamics simulations reveal two catalytic lysine residues. Although only a few site-directed mutagenesis based on conserved residues have been applied to probe the CAR catalytic properties,4,10,30 the enzymatic mechanism still remains unclear. In order to shed light on the structural dynamics of ATP and the substrate binding in the A domain, 300-ns MD runs by analyzing conformational changes were conducted. Benzoic acid was chosen as the model substrate. Taking advantage of the X-ray structures 5MSW and 5MSS, which correspond to adenylation and thiolation conformations of SrCAR, respectively, we constructed four models to mimic each catalytic steps of CAR (Scheme 1): (i) only ATP is positioned in the pocket before adenylation (dubbed “5MSW/ATP”), (ii) The substrate benzoic acid is adenylated with ATP, forming substrate-AMP complex (dubbed “5MSW/AMPSub”), (iii) The PPT arm was positioned to the active pocket, awaiting for thiolation (dubbed “5MSS/AMP-Sub/PPT”), and (iv) the substrate benzoic acid was covalently bonded with PPT after thiolation (dubbed “5MSS/AMP/PPT-Sub”). The SrCAR A domain active sites are mainly hydrophobic, existing many binding contacts with the substrate and ATP (Figure 2). Among them, the conserved A10 Lys629 (numbering according to SrCAR) (Figure S1) is a critical residue among the ANL members.12-19 It has been demonstrated that this residue can facilitate the orientation of substrate to catalytic adenylation partial reaction by providing favorable polar contacts that are critical for stabilizing transition states. In this study, during the 300 ns MD simulations, the ε-nitrogen atom (NZ) of Lys629 was frequently shown to interact with the ribose ring oxygen atom (O4´) and the bridging oxygen atoms (O5´ and O3A) of ATP before adenylation (Figure S4a). The average distances between NZ and O4´, O5´ and O3A are 3.52 ± 0.51 Å, 3.51 ± 0.48 Å and 3.64 ± 0.56 Å, respectively. After adenylation, the average distances of NZ-O4´ and NZ-O5´ are 3.01 ± 0.20 Å and 3.26 ± 0.26 Å, respectively, while an occasional hydrogen bond between the substrate carbonyl oxygen atom (O3A) and Lys629 at an average distance of 3.84 ± 0.36 Å was also observed (Figure S4b).

After activation, benzoic acid is transformed to a high energy complex (benzoic-5´-AMP, dubbed AMP-Bez), the A10 motif containing Lys629 is dragged out from the catalytic pocket and is solvent-exposed via the domain rearrangement (Figure 1), priming the enzyme for thiolation. MD simulations were then performed on the model “5MSS/AMP-Sub/PPT” and the “5MSS/AMP/PPTSub” for exploring the key residues dynamics before and after thioesterification. Strikingly, in the thiolation-forming conformation, a less conserved Lys528 residue (A8 motif, Figure S1) locates in a similar position regarding to Lys629 in the catalytic pocket (Figure S5), indicating that Lys528 is able to participate in the following thiolation step. The MD trajectories showed that Lys528 resembles Lys629, the NZ atom of Lys528 can form hydrogen bonds with the AMP-benzoic acid complex, including O4´, O5´ as well as O01 atoms (Figure 3a and Movie S2). The average distances are 3.54 ± 0.38 Å (NZ-O4´), 3.12 ± 0.21 Å (NZO5´) and 2.93 ± 0.19 Å (NZ-O01), respectively. In contrast, Lys528 in the model “5MSS/AMP/PPT-Sub” seems to make non-compact connections with AMP after thioesterification, the distances are relatively larger and variable (Figure 3b and Movie S3). Furthermore, the shape and size of the catalytic pocket in each step are strikingly different: the computed volumes of the catalytic pocket is enlarged from 375.94 ± 23 Å3 to 625.15 ± 27 Å3 after thiolation (Figure 4). Moreover, a larger pocket volume is also beneficial for AMP leaving the pocket after thiolation.

Figure 3. Traces of selected interactions monitored along the MD simulations of the systems regarding to the stages before (a) and after (b) thiolation. Distances between the nitrogen atom in the side chain of Lys528 and the oxygen atoms of the AMP-benzoic acid complex are represented as NZ-O4´, NZ-O5´, NZ-O01 and NZO3A.



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CH/π interaction with the β-methylene group against the adenine ring of AMP. The roles of these sites identified by IFP are consistent with the above conservational analysis (Table S2). In addition to the residue Lys528, some new residues (e.g. T265, S408, T505, etc.), which were not reported in previous studies, may play a key role in substrate recognition and product release.

Figure 4. Overlay of 500 snapshots derived from MD runs of A-T didomain before (a) and after (b) thioesterification. Each snapshot was extracted every 600 ps from the trajectory files. The PPT arm, AMP and benzoic acid moiety (Bez) are depicted by sticks. Practically, with the aim of elaborating the function of the two catalytic lysine residues Lys629 and Lys528, alanine substitution was introduced to the two sites, respectively. As a result, in comparison to wild-type (WT) SrCAR, the benzoic acid is reduced to the alcohol product with the assistance of E. coli endogenous alcohol reductase6,42,43 at a 94% conversion, while the two mutants did not show any activity for benzoic acid (Figure 5 and Figure S6). The site-directed mutagenesis experiment supports the essentiality of the two lysine residues Lys629 and Lys528 in the adenylation and thiolation states, respectively.

Figure 5. HPLC profiles of WT SrCAR, K528A and K629A. The original HPLC results and SDS-PAGE analysis are shown in Figure S6. Protein−ligand interaction fingerprint analysis (IFP) was carried out to explore the atomic details of active substrate−CAR interactions, including the active substrate in complex with AMP (model “5MSS/AMP-Sub/PPT”) and PPT (model “5MSS/AMP/PPT-Sub”), respectively (Figure 6). With regard to the AMP moiety, the purine forms hydrogen bonds with the main chain amides of G430 and G432, and the ribose hydroxyls interact with the side chains of D507, R522 and Y519, while the phosphate forms a hydrogen bond with T434. Y431, however, undergoes

Figure 6. Substrate binding modes and protein−ligand interaction fingerprint for 5MSS/AMP-Sub/PPT (a) and 5MSS/PPT-Sub/AMP (b). The IFP radar-plots depict the statistical protein−ligand interaction frequency obtained from the MD runs by using PLIP tool.44 A “switch” mechanism of the two lysine residues during the successive reactions. The crystal structures have clearly represented that the domain rearrangement results in Lys629 and Lys528 are always apart (Figure 2), one of the two lysine residues stays away from the active pocket when the other one in the pocket. In details, the distances between Lys629 and Lys528 in the static crystallographic structures 5MSW and 5MSS are 14.6 and 26.9 Å, respectively, while during MD simulations, the corresponding distances are 20.2 ± 0.6 and 24.2 ± 1.1 Å in the models “5MSW/AMP-Sub” and “5MSS/AMP-Sub/PPT”, respectively. Apparently, the two lysine residues are switched during the successive reactions: in the adenylation stage, Lys629 gathers ATP and carboxylate in the binding pocket by forming hydrogen bonds with the substrate carboxyl group and oxygen atoms of the ribose moiety of ATP. The substrate carboxyl group is then under a nucleophilic attack by the α-phosphorus atom of ATP, forming the pentacoordinated transition state (Figure 7). AMP-Bez complex and PPi are released through cleavage of the P-O bond. After adenylation step, the A-T didomain rotates (Figure 1), representing a closed rather than a stretched conformation (Figure S2c). In the following thiolation reaction, the interaction between Lys528 and the AMP-Bez complex stabilizes the complex and thus drives nucleophilic attack by the thiol group of the PPT arm. As such, the thiol group accepts a proton donated from the departing phosphate oxygen via the non-bridging phosphate oxygen. After the cleavage of the C-O bond, PPT-Bez intermediate is formed. The conformational change and the mechanism of adenylation and thiolation reactions are proposed in Figure 7.


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Asub

Asub

K528

Asub

K528

K528

K629

K629

K629

K629

N N H H H H H H H H Adenylation O O HO HO O O O P O PPi O P O PPi Ad Ad O O O O O O O

N H H H H Domain O HO rotation O O O P O Bez Ad O O

O

HO

K629

Ad

S

PPT

Asub

HO Ad

K528

N H H H H O O O O P O Bez O O S

T

PPT

Asub

K629

K528

N H H H H Thiolation O O O O P O Bez O O H

PPi

Bez

Bez

Asub K528

N H H

H AMP O

Bez S

T

PPT

T

Figure 7. The proposed “switch” mechanism of Lys629 and Lys528 in the adenylation and thiolation reactions. PPi represents pyrophosphate. Proposed reduction mechanism of the R domain. After thiolation, the thioester intermediate is shuttled into the R domain, where the intermediate and the cofactor NADPH are maintained by hydrogen bonds and hydrophobic interactions (Figure S7). Particularly, the R domain pocket bears a canonical tyrosinedependent catalytic triad Thr935-Tyr970-Lys974 (Figure S7a), which is shown at similar positions as reported in SDR family and analogues.20-24,45 In addition, a histidine residue (His900) located 3.5 Å from the thiol group was observed (PDB code 5MSV). In order to gain more insight, a model containing apoprotein, NADPH and thioester intermediate (dubbed “5MSV/NADPH/PPT-Sub”) was constructed based on 5MSV. Catalytically competent poses sampled along the MD simulations can be determined by the distance and the attack angle between nucleophile and electrophile.46,47 a relative populations of the catalytically competent conformations were also observed by evaluating the distance and the attack angle between NADPH and the substrate during the 300 ns unrestrained MD simulations (Figure S8a). In parallel, the four residues (Thr935, Tyr970, Lys974 and His900) maintained reasonable distances from the substrate carbonyl, NADPH ribo moiety as well as the thio group (Figure S8b). Moreover, it has been demonstrated that the essentiality of the catalytic triad Thr-Tyr-Lys and His900 by site-directed mutagenesis,4,30 supporting that the four residues are indispensable for the hydride transfer process. Although the hydride and proton transfer mechanism of CAR is still unclear, other representative NADPH-dependent enzymes, such as SDR family and HMG-CoA reductase, have already been investigated (Scheme S1). In SDR family, the catalytic triad Thr/Ser-Tyr-Lys can form a hydrogen bonding network that is crucial not only for stabilizing the transition state, but also for proton transfer (Scheme S1a). In contrast, a histidine residue is thought to be the proton donor for the CoA thiolate anion in the reduction catalyzed by HMG-CoA reductase (Scheme S1b).48 Considering that the R domain may adopt either of the two representative enzymatic proton transfer pathways, we constructed two models with respect to the proton transferred by the catalytic triad (Scheme 3a), and by the protonated histidine residue (Scheme 3b). (a)

(b)

PPT

PPT

NH

NH

N

S

S

His

H

H

H H

H 2N N

NADPH

O

O Thr

H

H

His

N

H H

NH

Tyr

Lys

O

NADPH

O

Thr

O

H O

OH

H

H

H 2N

O

O

N

O

O O

H

H O H OH

H

N

Tyr

Lys

Scheme 3. Two candidate proton transfer pathways constructed for the SrCAR R domain. The hydrogens and the reactive hydrides proposed to be participated in the proton relay are colored in blue and red, respectively. Quantum chemical calculations to investigate the proton and hydride transfer in the R domain. Consequently, it is of great interest to compare the two distinct models (Scheme 3) for catalyzing the hydride and proton transfer process. Moreover, it also remains elusive whether the proton and hydride are added via concerned or stepwise mechanism. We herein used the quantum chemical cluster approach, which is small enough to be calculated wholly using high-level QM methods, and which has already been demonstrated to be a successful methodology for probing the enzymatic mechanism,32,49,50 including the hydrogen transfer mechanism.22,27,46 The cluster model, which contains the cofactor NADPH that was abbreviated as a nicotinamide-ribofuranose complex, was extracted from the coordinates of an MD simulation. The side chains of the catalytic Thr935, Lys974, Tyr970 and His900 were truncated to ethanol, phenol, methylammonium, and protonated imidazole, respectively. The activated complex derived from the MD simulations, in which the coordinates of the truncation atoms are fixed, were then performed at the B3LYP+D3/6311++G(2d,p) (SMD, solvent=water)//B3LYP+D3/6-31G(d,p) (SMD, solvent=water) level of theory. The calculated energetic and geometric results for the path A (Scheme 3a) are detailed in Figure 8. The reaction is initiated by the hydride transfer step via a free energy barrier of 17.0 kcal/mol (TS1). As expected, Thr935 and Tyr970 form an oxyanion hole with distances of 1.763 and 1.610 Å (also see Figure S9), respectively, in the transition state TS1 to stabilize the developing negative charge on the thioester oxygen. The formed intermediated IM1 is a little bit less stable than Re, but it can be easily transferred to the slightly stable IM3 via stepwise proton transfer steps. The barriers for the proton transfer steps are quite small (TS2, TS3, and TS4) or even barrierless (TS3), implying the very facile hydrogen transfer process. With the assistance of a water molecule (Figure S10), the hydrogen atom from the hydroxyl group of the substrate moiety can be easily transferred to the sulfur atom via an accessible barrier of 21.5 kcal/mol (IM4→TS5). Notably, the direct hydrogen transfer without water molecule is excluded because of the high barrier (~27.1 kcal/mol, Figure S11). The water molecule, thus acts as reactant, proton shuttle, product, and catalyst during the reaction pathway, which functions in the stabilization of the transition states and intermediates. The overall reaction is exoergic by 7.4 kcal/mol, and the barrier for the rate-determining step is predicted to be 21.5 kcal/mol, which is kinetically and thermodynamically accessible for experiment under mild reaction conditions. We also explored a different reaction channel (path B, Figure S12), in which proton transfer occurs first followed by hydride shift. The energetics and geometric data in Figure S12 and Figure S13 suggested that it is less favorable than that depicted in Figure 8.


Journal of Chemical Information and Modeling PPT

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PPT

NH

N S 2.565 H H 1.295 O O 1.763 H 1.339 H O H 1.360 1.610 O H H2N 1.007 O N 1.753 H 0.986 O O 1.872 H 1.042 NH OH H NADPH

His

O Thr

0.995 O

O

Tyr

Lys

O

O

NADPH

Tyr

H

1.870 OH

1.041 NH

H

OH

NADPH

Lys

TS2

O H2N

O

Tyr

1.068 NH

H

Lys

2.592

H2N

1.007 H 1.647 O 1.244

NADPH

TS3

O

Tyr

NADPH

Lys

O

0.1 [3.0]

Re

IM1

TS2

N O

NADPH

0.981 O

H

PPT

NH

O Thr H2N N

H

Re

O

Tyr

1.044 NH Lys

NADPH

0.989 O

-0.2 [1.5]

-0.1 [1.7]

-0.1 [4.1]

0.1 [-0.4]

IM2

TS3

IM3

TS4

IM4

PPT His

Thr

Tyr

Lys

O O H2N N

H

H

O

Tyr

1.042 NH

IM1

Lys

NADPH

1.027 O

H2N N

1.559

H

1.800 OH

O O 1.775 H 0.972 H H O 2.383

O

O

H

H

O

Tyr

1.046 NH Lys

-7.4 [9.5]

NADPH

IM2

1.029 H 1.559 O 1.584 OH

H

His H

Thr

O

H 1.087 NH

IM3

Pr

PPT

NH S 2.574 H

H

Thr

H2O

N

His

O 1.803 H 1.016 H H O 1.611

Thr

PPT

NH N 2.310 S H

1.766

H

1.849 OH

-1.1 [-0.2]

NH

N S 2.314 H H 1.352 O O 1.676 H 1.103 H 1.458 O H 1.051 O

His

1.793

1.831 H OH

O

TS5

0.6 [1.9]

H

His

21.6 [23.6]

0.0 [0.0]

H2N

H 1.762

TS5

TS1

N S 2.666 H H 1.238 O O 1.836 H H 1.772 O 1.097 H 0.986 O

H 1.059 O H H 1.013

1.011 H 1.573 O 1.035 H 1.630 NH OH H

TS4

17.0 [20.1]

PPT

N

O

N

H 1.287 NH H

OH

S

H O 1.270 1.574

O Thr

O

N

1.865

H

O 1.779 H 0.972 H H O 2.369

Thr

1.198 O N H 1.241 O O 1.655 H

1.755

H

His O

O 1.787 H 0.971 H H O 2.500

Thr H2N

H

NH

PPT

NH N 2.498 S H

His

S 2.280 H

1.208 O N

TS1 G E]

His H

O 1.725 H 1.214 O H

H2N

PPT

NH N

O

H

PPT

NH N S 2.338 H

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Tyr

Lys

1.862

NH 2.362

S

N

H H 1.409 O H O O 0.972 H H 1.762 O H2N 2.293 O 0.996 N H 1.680 O O 1.024 H 1.694 NH OH H NADPH

His

Thr

Tyr

Lys

IM4

Figure 8. Free energy profile for the reduction of benzoic acid by SrCAR at the B3LYP+D3/6-311++G(2d,p) (SMD, solvent=water)//B3LYP+D3/6-31G(d,p) (SMD, solvent=water) level of theory. Values in kcal/mol are free energies and energies (in the brackets), respectively. NADPH ribose, and Thr935 along with Tyr970 are proposed to form the oxyanion hole that assists in reduction of the thioester. On the other hand, the reaction mechanism on the basis of Particularly, His900 helps to stabilize the TSs and decrease the cluster model in Scheme 3b (path C) was also examined. However, energy barriers. The results indicate that the hydride and proton as detailed in Figure S14 and Figure S15, the free energy barrier transfer occur via a stepwise mechanism, and the hydride transfer for path C is as high as 25.4 kcal/mol, which is higher than that of is proposed to be the rate-determining step for the overall reaction. path A based on Scheme 3a (21.5 kcal/mol, Figure 8). This Protein engineering has proven to be a powerful mean to suggests that path C is also disfavored. Moreover, conservation remarkably improve the enzymatic performance,51-53 whereas such analysis indicates that the histidine residue is less conserved efforts in CARs are rather scarce. Taking the advantage of rational compared to the catalytic triad (Figure S16), indicating that His900 design or directed evolution, it can be expected that CAR enzyme could not a catalytic site. Perhaps His900 is achieved by natural will be more attractive in organic chemistry and biotechnology. evolution due to the stabilization of the TS rather than to proton Upon detailing the conformational dynamics and reaction transfer in the R domain of SrCAR. Here, we probe the energetics of reaction mechanism with a model of the SrCAR active sites mechanism of SrCAR, the key residues uncovered in this study may consisting of the four residues proposed to be implicated in the provide guidance for future protein engineering. reaction mechanism, and gained insight into a deeper understanding of CAR-mediated catalysis, in particular to provide an explanation MATERIALS AND METHODS for the proton transferation. Model generation and Molecular dynamics (MD) simulations. Schrodinger Maestro software54 was used to prepare protein and CONCLUSIONS AND PERSPECTIVES ligand structures. Crystallographic structures of SrCAR served as starting geometries: PDB codes 5MSW and 5MSS are used for With the aim of seeking a deeper understanding of the adenylation and thiolation modelling, respectively, while 5MSV conformational dynamics and catalytic mechanisms of CAR, we are employed for R-T reduction modelling. Missing residues in investigated the vital role played by Lys629 and Lys528 via MD simulations, which were further confirmed by site-directed 5MSW, 5MSS and 5MSV (Figure S17) were completed by Modeller 9.19,55 using 5MST, 5MSR and 5MSP as templates. In mutagenesis. The rotation of the A-T didomain is essential for the order to assemble the substrate benzoic acid conformation, the pose enzyme to switch the proper lysine residues for the successive of benzoic acid in 5MSD was superimposed into 5MSW, to form adenylation and thiolation reactions. The switch mechanism of ATP-Bez binary complex (in “5MSW/AMP-Sub” model). The Lys629 and Lys528 is first reported in CARs. QM calculations were pose of phosphopantetheine complex in 3NYQ was superimposed further performed to study the reduction process, it can be into 5MSS, to form PPT arm (in “5MSS/AMP-Sub/PPT” model) concluded that the SrCAR R domain employs a canonical Thr-Tyrand PPT-Bez complex (in “5MSS/AMP/PPT-Sub” model), Lys catalytic triad in associated with a non-conserved histidine respectively. In the same way, the thioester geometry of the residue: Lys974 is permanently hydrogen bonded to the cofactor


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phosphopantetheine complex in 1W6U was used to generate PPTBez complex in “5MSV/NADPH/PPT-Sub” model. The phosphate ion of phosphopantetheinyl group was bonded to hydroxyl oxygen atom of Ser702. Protonation of each apo-protein was performed at pH 7.4 (to mimic the the experimental conditions) using H++ webserver.56 Lys and Arg were assigned as being positively charged, whereas Glu and Asp were negatively charged. The His900 has a proton on the Nδ position to evaluate whether it is involved in the hydrogen transfer, while other His residues have a proton on the Nε position. The Amber ff14SB force field57 was used to describe the protein in all of the MD runs. Each system was solvated in a cuboid box with a 10 Å buffer of TIP3P water molecules58. Charges and parameters for substrate complexes and PPT arm (listed in Table S4) were generated with the Antechamber (AM1-BCC)59 using the amber GAFF force field.60 The Ser702 portion of the PPT molecule uses standard serine parameters, whereas the PPT portion adopts GAFFderived parameters (Table S4). For cofactors ATP and NADPH, the parameters and charges were provided by the Amber Parameter Database (http://sites.pharmacy.manchester.ac.uk/bryce/amber).61,62 Eexplicit counterions (Na+ and Cl−) were added to neutralize the total charge of the systems. In order to remove poor contacts and relax the systems, minimizations were carried out by using 5,000 steps of steepest conjugate and 5,000 steps of conjugate gradient. Thereafter, the systems were heated from 0 to 303 K under NVT conditions for 50 ps. Subsequently, the systems were maintained for 50 ps of density equilibration under NPT conditions at constant temperature of 303 K and pressure of 1.0 atm using Langevinthermostat (ntt=3) with a collision frequency of 2 ps-1 and pressure relaxation time of 1 ps. A weak restraint of 10 kcal mol-1 Å-2 on the protein residues were carried out during the heating and density equilibrations. After removal of all restraints, the systems were further equilibrated for 10 ns to get well settled pressure and temperature. After that, a productive MD run of 300 ns was performed for each system. During all MD simulations, SHAKE algorithm63 was applied on all bonds with hydrogen. The trajectory file was written every 100 steps with a MD time-step of 2 fs. All above MD runs were carried out using the GPU version of Amber 2016 (comprised of AmberTools16 and Amber16).64 The binding site volume was calculated via POVME program.65 The convergence of all the MD simulations were evaluated by rootmean-square deviation (RMSD) analysis (Figure S18). Site-directed mutagenesis and activity assay. The gene of wildtype (WT) SrCAR (GenBank: WP_007468889) was synthesized and inserted into Nde I and Xho I sites of vector pET24a. The mutants of SrCAR were constructed using the megaprimer approach66 with PrimeSTAR DNA polymerase. Primers were listed in Table S5. The PCR conditions for short fragment: 98 oC for 2 min, (98 °C for 10 s, 55 °C for 15 s, 72 °C for 15 s,) ×26 cycles, 72 °C for 5 min. For mega-PCR: 98 °C for 2 min, (98 °C for 10 s, 60 °C for 15 s, 72 °C for 3 min,) ×26 cycles, 72 °C for 5 min. The PCR products were analyzed on agarose gel by electrophoresis, then 0.5 μL Dpn I was added in 10 μL PCR reaction mixture and the digestion was carried out at 37 °C for 2 h. After it, the PCR products were directly transformed into electro-competent E. coli BAP167 to create the SrCAR mutants. To perform the activity assay, the E. coli BAP1 strains harboring pET24a, pET24a-WT, pET24a-K629A and pET24aK528A were inoculated into test-tubes containing LB (5 mL) and Kanmycin (50 μg/mL), respectively, for 8 h (37 °C, 220 rpm). This preculture was inoculated into 100 mL TB medium containing Kanamycin (50 μg/mL) and was incubated at 37 °C until the OD600 reached 0.8-0.9, and then Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM and was incubated for additional 16 h. Cells were collected by centrifugation,

washed with phosphate buffer (50 mM, pH 7.4) and suspended again in phosphate buffer. A part of the cells were lysed by sonication and were subjected to the SDS-PAGE analysis. The other cells were then transferred into a 50 mL Erlenmryer flask. In continuation, 22.2 mM Glucose and the substrate benzoic acid (10 mM) were added. The reaction was carried out at 30 °C, 220 rpm for 24 h, and terminated by adjusting the pH to 2-3 with 1 M HCl solution. The aqueous phase was centrifugation at 12000 rpm for 10 min and analyzed by HPLC equipped with an Agilent SB-Aq C18 column (4.6 mm×250 mm×5 μm) with a flow rate of 1 mL/min (the ratio of acetonitrile to 0.1% TFA is 4:6) at 220 nm. Benzoic acid and benzyl alcohol were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China) and Shanghai Macklin Biochemical Co., Ltd (Shanghai, China), respectively. All the assays were performed in triplicate. Quantum mechanics (QM) calculations. The starting geometry was obtained after equilibration of the system, which depicts a Cα RMSD of 0.384 Å, compared to the original X-ray structure 5MSV (Figure S19). Geometry optimizations without symmetry restrictions were carried out for all molecules using the Gaussian 09 program,68 with the B3LYP69,70 functional with 6-31G(d,p)71-75 basis set and dispersion correction by Grimme76-77 with BeckeJohnson damping D3 and solvation effects accounted by the SMD78 model using experimental used solvent water, which is termed as B3LYP+D3/6-31G(d,p) (SMD, solvent=water). It should be emphasized that the terminal atoms of the truncated residues have been fixed to simulate the reasonable real pocket environment. Frequency results were examined to confirm the stationary points as transition states (only one imaginary frequency) or minima (no imaginary frequencies), as well as used to obtain the zero-point energy-corrected enthalpies and free energies at 298.15 K and 1 atm. IRC (intrinsic reaction coordinate)79,80 analysis were performed to evaluate the correct connections between the transition states and corresponding minima if necessary (Figure S20). The energetic were further improved by using the larger basis set 6311++G(2d,p)81, which denoted as the B3LYP+D3/6-311++G(2d,p) (SMD, solvent=water)//B3LYP+D3/6-31G(d,p) (SMD, solvent=water) level; electronic energies without zero point corrections were also given for reference in the related schemes. Coordinates and energies (in Figure 8) of the calculated structures were listed in Table S6.

Accession codes In this study, we have undertaken the reaction mechanism of Segniliparus rugosus carboxylic acid reductase (Uniprot accession code E5XP76, as well as PDB accession codes 5MSW, 5MSS and 5MSV).

ASSOCIATED CONTENT Supporting Information. Supplementary schemes, figures, movies, tables and QM calculation data.

AUTHOR INFORMATION * Corresponding Author [email protected] # Authors contribute equally to this work. Notes The authors declare no competing financial interests.



ACKNOWLEDGMENT We acknowledge Prof. Manfred T. Reetz for commenting on the manuscript. Z.S thanks CAS Pioneer Hundred Talents Program (No. 2016-053), the Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-212), and the Key Projects in the Tianjin Science & Technology Pillar Program (No. 15PTCYSY00020). G.Q thanks Science & Technology Foundation for Selected overseas Chinese scholar of Tianjin (2017) for generous support. L.Z thanks the National Natural Science Foundation of China (No. 21703099), Natural Science Foundation of Jiangsu Province for Youth (No. BK20170964), the financial support from Nanjing Tech University (No. 39837123) and SICAM Fellowship from Jiangsu National Synergetic Innovation Center for Advanced Materials, and the High Performance Computing Center of Nanjing Tech University for supporting the computational resource.

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