Catalytic Roles of Histidine and Arginine in Pyruvate Class II Aldolase

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Catalytic Roles of Histidine and Arginine in Pyruvate Class II Aldolase: A Perspective from QM/MM Metadynamics Gou-Tao Huang, and Jen-Shiang K. Yu ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b03398 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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ACS Catalysis

Catalytic Roles of Histidine and Arginine in Pyruvate Class II Aldolase: A Perspective from QM/MM Metadynamics Gou-Tao Huang† and Jen-Shiang K. Yu∗,†,‡,¶ †

Department of Biological Science and Technology, ‡ Institute of Bioinformatics and

Systems Biology, and ¶ Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu City 300, Taiwan E-mail: [email protected]

Abstract The retro-aldol reaction catalyzed by pyruvate class II aldolase is investigated with QM/MM metadynamics; this enzyme transforms the substrate of 4-hydroxy-2-ketoacid into pyruvate and aldehyde through the aldol cleavage. The hydroxyl group of the substrate is deprotonated by His45 with the aid of the metal-bound water, while the metal-bound hydroxide proposed in the literature is observed as a transient species. The deprotonation appears to enhance substrate binding between the deprotonated substrate and the active site. The reactive alkoxide is further stabilized by the salt bridge of Arg70-Asp42, facilitating the following aldol cleavage. The simulations show that the C−C bond cleavage is the rate-determining step, and the calculated barrier of approximately 14 kcal mol−1 agrees reasonably with experimental data. ∗

To whom correspondence should be addressed National Chiao Tung University ‡ National Chiao Tung University ¶ National Chiao Tung University †

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Keywords aldol cleavage, salt bridge, aldolase, pyruvate, QM/MM, metadynamics

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Text

The formation of carbon-carbon bonds is important to construct the backbone of organic molecules. The aldol condensation is one of the well-known reactions for the C−C bond formation, where two carbonyl molecules are coupled to one aldol product and its reverse process is the aldol cleavage (or a retro-aldol reaction). The aldol-cleavage/condensation reactions are usually reversible in the presence of an acid or a base, and can be achieved through biocatalytic processes. 1–3 In enzyme catalysis, the aldol reaction can proceed through two known mechanisms of 1) the involvement of the Schiff base 4 produced from the reaction of lysine with the carbonyl group of a substrate, and of 2) the participation of a divalent metal ion as a cofactor. 5 Due to different catalytic mechanisms, the former are referred to as class I aldolases, and the latter as class II aldolases. In the class II aldolases (a.k.a. metalloaldolases), the divalent metal acts as an electron sink, which stabilizes the negative charge of the enolate formed during catalysis. The pyruvate aldolase that is one of the class II aldolases utilizes pyruvate as the nucleophilic substrate, which would react with an electrophilic aldehyde through the aldol condensation. HpaI (also known as HpcH) is a class II pyruvate aldolase, featuring the divalent metal coordinated octahedrally with the carboxylic residues of Glu/Asp, bidentate substrates, and water molecules. 6 According to X-ray crystal structures, kinetic analyses, and mutation experiments, Arg70 and Asp42 have significant effects on the catalytic activity. 7–10 Figure 1 shows the mechanism of the retroaldol reaction proposed by Seah and coworkers. 10 The key metal hydroxide is first formed through the proton abstraction by His45 (preR → R1), and then abstracts the hydroxyl proton of the substrate (R1 → R2) to promote the aldol cleavage (R2 → P1). After the aldehyde product leaves the active site, the initial state is regenerated through the proton 2


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transfer among the protonated His45, W1 and the pyruvate enolate with participation of an additional water molecule (P2 → P3 → preR). It has been reported that His45 was involved for substrate binding and proton abstraction. 7 Arg70 forms a salt bridge with the neighboring Asp42 residue, and the absence of either residue of Arg70 or Asp42 leads to the loss of the catalytic activity. 7,10 In the present work, the QM/MM metadynamics simulation is used to explore the deprotonation and the aldol cleavage, marked in the gray background region of Figure 1. The catalytic roles of His45, the metal-bound water/hydroxide, and the salt bridge Arg70-Asp42 are to be elucidated. preR reactant state H N

Asp42

N d3

W3 H O

W2 H O H

H

O

O Asp175

NH

W1 H H d4 O

d2

H d1 O

4

O 1

O

3

2

dc

H d4

d5

O1

d7

M2+

O

R H

W1OH H HN H O d2 H

NH

M

4 3

2

dc

d3

H

NH

W1 H O H d2

TS2H

R H

Asp42 O

Arg70

N

d4

c

O

His45

NH H

O

2+

H N

O

d1 O

TS1H

O O 4-hyroxy-2-ketoacid

O

Asp42 O

Arg70

N

d3

NH H

HN H

dO

His45

O

Arg70

His45

R2 deprotonated reactant state

R1 H N

d1

O

c

4

O

M2+

1

O

1

O

O

NH H

HN H

3

2

dc

R H

c

O

Glu149 d H1 d H2

pyruvate + H2O

d1 d4

d2 d3

TSC

substrate

H N His45

N

H H O W1

O NH H

HN H H

O

Asp42

H N

O

Arg70 NH

H

His45

N H

Arg70 NH H

W1 O H

3 2

O

1

2

O pyruvate P3

O

M2+ O

O

Arg70 NH

N H

H

O

O H

O

M2+

His45

W1

NH H

HN H

Asp42

H N

Asp42

3

O H

O O NH H

HN H

O H

M

3

H2O

O

O

H

R

1

O

P2

4

O

2+

1

R

H

2

O pyruvate enolate product state P1

Figure 1: Proposed mechanism for the retro-aldol reaction. The octahedral coordination is illustrated with red dashed lines. Mg2+ and (R)-4-hydroxy-2-oxopentanoate (HPA) were chosen as the divalent metal cofactor and the substrate in the simulation, respectively. The quantum mechanical region composed of 104 QM atoms (Mg2+ , HPA, W1, W2, W3, and nine residues of Asp42, His45, Arg70, Glu44 ,Gln147, Glu149, Asp175, Asp84, and Val118’, shown in Figure S9) 3


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was computed at the ωB97X-D 11 level using Gaussian 09 package. 12 Amino acids and water molecules in the non-QM region were treated by molecular mechanics (MM) with the force fields of AMBER-99SB and TIP3P, respectively. 13,14 The well-tempered metadynamics was run using the Gromcas 4.5.5 package 15 combined with the PLUMED 2.1.1 software. 16–21 Computational details of the simulations are provided in ESI. WTMetaD-H and WTMeta-CC denote the metadynamics simulations with respect to the deprotonation and the aldol cleavage, respectively. Three sets of collective variables (CVs) were defined by dC−C , dH1 ≡ d1 − d2 and dH2 ≡ d4 − d3 , shown in Figure 1. The CV of dH2 was biased in WTMetaD-H, while the WTMetaD-CC simulation exerts the bias potential to the C3−C4 bond, namely dC−C . Preliminary simulations in the product state of P1 showed that without constraint to the C3−C4 bond, acetaldehyde departs away from the metal-bound pyruvate. This poor binding between aldehyde and the active-site pocket is also reflected by the experimentally observed broad specificity for aldehyde substrates with a rapid-equilibrium random-order mechanism. 9 For the time-consuming simulation, it would take a relatively longer time to return to the bound product state once aldehyde leaves the active site. To accelerate the sampling distributions during the bond-breaking process, the upper wall was set at 3.3 ˚ A for dC−C . Although the dissociative product state larger than the upper wall was little populated, the bond-breaking region (dC−C at TSc : 2.4 ˚ A) can be well illustrated from the computed free energy surface. Figure 2a shows the free energy landscape of the deprotonation. Two main wells corresponding to preR and R2 are observed, and the two states exist at equilibrium while the metal-bound hydroxide (R1) is a transient species during the deprotonation process. Also, the sampling numbers of R1 and R3 are small in the equilibrated simulations (Figures S5 and S10-12). Mechanistically, the proton abstraction of W1 by His45 is followed by the deprotonation of the hydroxyl group, and this sequential process is confirmed by the WTMetaD-H simulation. The low barrier of 5.7 kcal mol−1 indicates that the formation of the alkoxide is facile in the assistance of His45. The comparison of the bond lengths between

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the preR and R2 states in Figures S10-S12 shows that after the deprotonation the threes bonds of dO−O1 , d5 and d7 with respect to the substrate are shortened, which implies that His45 concerning the deprotonation affects substrate binding, in consistence with the experimental result that the substrate dissociation constants increased in the mutant enzymes of H45A and H45Q. 8 This can be anticipated because a negatively-charged species binds strongly with the positively-charged active site composed of the central divalent metal ion and Arg70. In regard to the His45 position in the R2 state, the protonated imidazole prefers interacting with W3, as observed in the crystal structure (Figure S6). The short-lived R1 state is formed only when His45 approaches W1 (at 10 ps in Figure S10b). The structural comparison between the enzyme and the gas-phase geometry based on the octahedral framework (Figure S2) reveals that the existence of Arg70 facilitates the alkoxide formation in the R2 state. However, according to the pKa prediction, the guanidinium group of Arg70 (pKa : 12) would be deprotonated to a neutral form when reacting with the alkoxide (pKa : 16, for ROH). This can be explained by the accompanying effect of the neighboring Asp42 residue: the salt bridge composed of guanidinium and carboxylate can stabilize the alkoxide, which is demonstrated by the model system (Figure S3). In addition to the known function 10 for maintaining the active site, Asp42 is involved in the stabilization of the alkoxide. Consequently, the deprotonated substrate is the dominant reactant species after the deprotonation whereas the neutral hydroxyl of R1 and R3 is of less significance (Figures S10b and S12b). If Arg70 was replaced by alanine, the catalytic activity was lost. 10 In the crystal structure of this mutant enzyme, the space of the Arg residue was occupied by water molecules (Figure S6c). The loss of the catalytic activity might be because the alkoxide is protonated by water with a similar pKa value, which is not able to effectively stabilize the alkoxide compared to the salt bridge. The simulations demonstrate that His45 is relevant to both deprotonation and substrate binding while Arg70-Asp42 is responsible for stabilizing the deprotonated substrate. In enzyme catalysis, amide NH bonds can act as an oxyanion hole to stabilize an alkox-

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ACS Catalysis

a) WTMetaD-H 25

R2 (1.4) 0

10

Arg70

His45

His45 1.80

15

TS2H (4.9) R1 TS1H (4.6) (5.7)

preR (0.0)

Arg70

20 kcal mol!1

dH1 (Å)

1

1.26

1.60

1.57 1.13

W3

1.98

2.11

2.13

3.57

W3

1.24

2.44

1.57

1.23 2.05

1.55

1.10

0.96 1.64

2.33 2.22

2.40

5

!1

0 !2

0

!1

1

2

TS1H

dH2 (Å)

TS2H

b) WTMetaD-CC 25 P1 20 TSC (13.7)

15

R2 (0.0)

10

Arg70

His45

kcal mol!1

1

dH1 (Å)

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

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1.81

1.07

1.57

W3

2.80

1.84 2.39 0.98 1.73 2.19 2.12 1.98

5

0

2

dC!C (Å)

3

0

TSC

Figure 2: Free energy landscapes for a) deprotonation and b) C−C bond cleavage. The energies (in kcal mol−1 ) are given in parentheses. The snapshot structures of the transition states are depicted, and the selected bond lengths are listed in ˚ A.

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ide. 22,23 In the case of the HpaI enzyme, such an oxyanion hole is composed of the salt bridge and the metal-bound water. The resulting alkoxide stabilized by the oxyanion hole is a reactive species for the subsequent aldol cleavage. Figure 2b shows the free energy surface of the aldol cleavage. The free energy of activation is 13.7 kcal mol−1 in the WTMeta-CC simulation. At TSC , the C3−C4 distance of ca. 2.4 ˚ A is slightly longer than the calculated bond length of 2.1−2.3 ˚ A in the computational literature of aldol-cleavage/condensation reactions. 24–27 As for the last step (P2 → P3), kinetic analyses 10 have indicated that the C3 proton exchange was not the rate-determining step. The mechanism of this proton transfer resembles to that of the deprotonation so that the barrier is expected to be comparable. The reaction rate is thus dominated by the C−C bond cleavage. In experiments, the measured rate constant (kcat ) was 353 s−1 using Co2+ as the cofactor although kcat with Mg2+ was not reported. 10 Derived from the kinetic data, the barrier height is about 14 kcal mol−1 according to the Eyring-Polanyi equation. Furthermore, the Mg2+ -dependent HMG/CHA aldolases with similar active-site architectures displayed a rate constant of ca. 15 s−1 , which corresponds to a free energy barrier of 16 kcal mol−1 . 28 The calculated barrier of 13.7 kcal mol−1 from the metadynamics simulation agrees reasonably with the experimental data, which indicates that the C−C bond breaking process is the rate-limiting step. In conclusion, the QM/MM simulations demonstrates the catalytic roles of His45 and Arg70 in the retro-aldol reaction. His45 is involved in the deprotonation and substrate binding, and the metal-bound hydroxide is a transient species during the deprotonation. Arg70 forms a salt bridge with Asp42, facilitating the formation of the reactive alkoxide. The barrier for the deprotonation (preR → R1 → R2) is lower than that for the aldol cleavage (R2 → P1), which indicates that the latter step is rate-determining. The estimated free energy of activation (ca. 14 kcal mol−1 ) agrees with experimental data. The observed solvent isotope effect 10 might be the result of a secondary isotope effect caused by the H/D exchange in the metal-bound water as well as Arg70. These theoretical perspectives could be beneficial to the development and design of the aldol reaction in enzymatic catalysis.

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Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Energetics and geometries in the model reaction. Optimized octahedral structures mimicking the active site of the enzyme. Effects of the salt bridge on the hydroxyl group. Details of computational setups in classic molecular dynamics, QM/MM simulation and QM/MM metadynamics. Author Contributions All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest.

Acknowledgement The authors are indebted to the Ministry of Science and Technology, Taiwan, for financial support under Grants MOST 106-2113-M-009-018-MY3, and the ”Center for Bioinformatics Research of Aiming for the Top University Program” of NCTU and MoE, Taiwan. G.T.H. acknowledges the postdoctoral fellowship from MOST 105-2811-M-009-001 and 106-2811-M009-040.

References (1) Trost, B. M.; Brindle, C. S. Chem. Soc. Rev. 2010, 39, 1600–1632 (2) Windle, C. L.; M¨ uller, M.; Nelson, A.; Berry, A. Curr. Opin. Chem. Biol. 2014, 19, 25–33 (3) Miao, Y.; Rahimi, M.; Geertsema, E. M.; Poelarends, G. J. Curr. Opin. Chem. Biol. 2015, 25, 115–123 (4) Choi, K. H.; Shi, J.; Hopkins, C. E.; Tolan, D. R.; Allen, K. N. Biochemistry 2001, 40, 13868–13875 (5) Daniel, D. E.; Horecker, B. L. Adv. Enzym. Relat. Areas Mol. Biol. 1968, 31, 125–181 8


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(6) Rea, D.; F¨ ulöp, V.; Bugg, T. D. H.; Roper, D. I. J. Mol. Biol. 2007, 373, 866–876 (7) Wang, W.; Seah, S. Y. K. Biochemistry 2005, 44, 9447–9455 (8) Wang, W.; Seah, S. Y. K. FEBS Lett. 2008, 582, 3385–3388 (9) Wang, W.; Baker, P.; Seah, S. Y. K. Biochemistry 2010, 49, 3774–3782 (10) Coincon, M.; Wang, W.; Sygusch, J.; Seah, S. Y. K. J. Biol. Chem. 2012, 287, 36208– 36221 (11) Chai, J.-D.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2008, 10, 6615–6620 (12) Frisch, M. J. et al. Gaussian 09 Revision D.01. Gaussian Inc. Wallingford CT 2009 (13) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Proteins: Struct., Funct., Bioinf. 2006, 65, 712–725 (14) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926–935 (15) Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E. Bioinformatics 2013, 29, 845–854 (16) Barducci, A.; Bussi, G.; Parrinello, M. Phys. Rev. Lett. 2008, 100, 1–4 (17) Laio, A.; Parrinello, M. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12562–12566 (18) Barducci, A.; Bonomi, M.; Parrinello, M. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2011, 1, 826–843 (19) Tribello, G. a.; Bonomi, M.; Branduardi, D.; Camilloni, C.; Bussi, G. Comput. Phys. Commun. 2014, 185, 604–613 (20) Bonomi, M.; Barducci, A.; Parrinello, M. J. Comput. Chem. 2009, 30, 1615–1621 9


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(21) Tiwary, P.; Parrinello, M. J. Phys. Chem. B 2015, 119, 736–742 (22) Simón, L.; Goodman, J. M. J. Org. Chem. 2010, 75, 1831–1840 (23) Richter, F. et al. J. Am. Chem. Soc. 2012, 134, 16197–16206 (24) Migues, A. N.; Vaitheeswaran, S.; Auerbach, S. M. J. Phys. Chem. C 2014, 118, 20283–20290 (25) An, W. Phys. Chem. Chem. Phys. 2015, 17, 22529–22532 (26) Zhang, X.; Houk, K. N. J. Org. Chem. 2005, 70, 9712–9716 (27) Zhang, S.; Deng, Z.-Q. Org. Biomol. Chem. 2016, 4, 1166–1169 (28) Wang, W.; Mazurkewich, S.; Kimber, M. S.; Seah, S. Y. K. J. Biol. Chem. 2010, 285, 36608–36615

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Graphical TOC Entry H N His45 N H His45 relevant to deprotonation H and subtrate binding O H O Asp175

Asp42 O

Arg70

O

NH

W1 H O H

O O

M2+ O

O

O Glu149

O

oxyanion hole to stabillize alkoxide

NH H

HN H

R H

O

M2+ = divalent metal ion (Mg2+, Co2+, Mn2+ ...)

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Catalytic Roles of Histidine and Arginine in Pyruvate Class II Aldolase

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