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Jitonnom, J., Mujika, JI, Van Der Kamp, MW, & Mulholland, AJ (2017)

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Jitonnom, J., Mujika, JI, Van Der Kamp, MW, & Mulholland, AJ (2017) Jitonnom, J., Mujika, J. I., van der Kamp, M. W., & Mulholland, A. J. (2017). Quantum mechanics/molecular mechanics simulations identify the ring-opening mechanism of creatininase. Biochemistry, 56(48), 6377-6388. https://doi.org/10.1021/acs.biochem.7b01032 Peer reviewed version Link to published version (if available): 10.1021/acs.biochem.7b01032 Link to publication record in Explore Bristol Research PDF-document This is the author accepted manuscript (AAM). The final published version (version of record) is available online via ACS at http://pubs.acs.org/doi/10.1021/acs.biochem.7b01032. Please refer to any applicable terms of use of the publisher. University of Bristol - Explore Bristol Research General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ 1 QM/MM Simulations Identify the Ring-Opening Mechanism of 2 Creatininase 3 4 Jitrayut Jitonnom*†, Jon I. Mujika‡, Marc W. van der Kamp‖,§, and Adrian J. 5 Mulholland§ 6 7 † Division of Chemistry, School of Science, University of Phayao, Phayao, 56000 Thailand 8 ‡ Kimika Fakultatea, Euskal Herriko Unibertsitatea UPV/EHU, and Donostia International 9 Physics Center (DIPC), P.K. 1072, 20080 Donostia, Euskadi, Spain 10 ‖ School of Biochemistry, Biomedical Sciences Building, University Walk, University of 11 Bristol, BS8 1TD, U.K. 12 § Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol 13 BS8 1TS, U.K. 14 15 16 17 18 19 20 21 To whom correspondence should be addressed: J.J.: Tel: +66 (0) 5446 6666 ext. 1834; Fax: 22 +66 (0) 5446 6664. E-mail: [email protected] 23 24 25 Abstract 26 Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) 27 to creatine via a two-step mechanism: water-addition followed by ring-opening. Water- 28 addition is common to other known cyclic amidohydrolases, but the precise mechanism for 29 ring-opening is still under debate. The proton donor in this step is either His178, or a water 30 molecule bound to one of metal ions, and the roles of His178 and Glu122 are unclear. Here, 31 the two possible reaction pathways have been fully examined by means of combined 32 quantum mechanics/molecular mechanics simulations at the SCC-DFTB/CHARMM22 level 33 of theory. The results indicate that His178 is the main catalytic residue for the whole reaction 34 and explain its role as proton shuttle during the ring-opening step. In the first step, His178 35 provides electrostatic stabilization to the gem-diolate tetrahedral intermediate. In the second 36 step, His178 abstracts the hydroxyl proton of the intermediate and delivers it to the cyclic 37 amide nitrogen, leading to ring-opening. The latter is the rate-limiting step with a free energy 38 barrier of 18.5 kcal/mol, in agreement with the experiment. We find that Glu122 must be 39 protonated during the enzyme reaction, so that it can form a stable hydrogen bond with its 40 neighbouring water molecule. Simulations of the mutant E122Q showed that this replacement 41 disrupts the H-bond network formed by three conserved residues (Glu34, Ser78, and Glu122) 42 and water, increasing the energy barrier. Our computational studies provide a comprehensive 43 explanation for previous structural and kinetic observations, including why H178A causes a 44 complete loss of activity but E122Q does not. 45 46 Keywords: QM/MM; creatininase, enzyme catalysis; SCC-DFTB; zinc metalloenzyme; free 47 energy 48 49 50 1. Introduction 51 Creatininase (creatinine amidohydrolase; EC 3.5.2.10), belonging to a member of the 52 urease-related amidohydrolase superfamily 1, 2, catalyzes the reversible interconversion of 53 creatinine to creatine (see Scheme 1), specifically acting on carbon-nitrogen bonds in cyclic 54 amides, like dihydroorotase 3 and other cyclic amidohydrolases 4. The enzyme plays a key 55 role in the bacterial degradation of creatinine 5 and participates in arginine and proline 56 metabolism 6. With growing industrial demand, considerable efforts have been made for 57 decades to characterize the properties of creatininase from various microorganisms, including 58 the bacterial enzyme from Pseudomonas putida 2, 7-12. Since the first X-ray crystal structure of 59 creatininase was reported in late 2002 by Beuth et al. 9, several structures have been 60 characterized to understand the structure and function of the enzyme 9-12. The structures 61 showed that creatininase has a unique structural fold (βα)4 compared to that of other members 62 of urease-related amidohydrolase superfamily 2, containing a binuclear metal center in each 63 subunit. Usually, two zinc ions are located at the M1 and M2 sites (hereafter called Zn1 and 64 Zn2, respectively) within the active site, ligated by five conserved amino acid residues 65 (Glu34, His36, Asp45, His120, Glu183) and two water molecules 10 (see Scheme 2). The two 66 zinc ions are bridged by a bidentate interaction with Asp45, which is functionally equivalent 67 to a carboxylated lysine residue found in related amidohydrolases, and by a zinc-bound water 68 molecule that is activated as a hydroxide ion during catalysis. 69 70 Scheme 1: Creatininase-catalyzed ring-opening reaction 71 72 Scheme 2: Two previously proposed reaction mechanism for creatinine hydrolysis catalyzed by creatininase 73 10 74 Path I: Histidine-promoted ring-opening pathway (Beuth et al.) His120 His120 His120 HN Asp45 H Asp45 H Asp45 H N HN HN His36 N N N C N His36 N C His36 C Glu34 Glu34 O O Glu34 O O O O O N O O N C N C O C O O O Zn2+ O 2+ Zn2+ C Glu183 O 2+ 2+ C Glu183 Zn1 Zn2 C Glu183 ZZnn1 Zn1 ZZnn2 O O Wat2 O Wat1 O O 1 w1 (1) water-adding step H O H O H O O O NH (2) ring-opening step O O Hw1 NH NH O H O H H H N O H 1 N NH N C 5 2 NH C N C H Glu122 O(H) 4 3 Glu122 O(H) N N His178 Glu122 O(H) N His178 His178 NH NH NH TI EP 75 ES 76 Path II: Water-promoted ring-opening pathway (Yoshimoto et al.) 12 Asp45 H Asp45 H Glu34 N Asp45 H Glu34 N His36 Glu34 N His36 C C C His36 His120 C O O His120 C O O O C O O O N His120 O N O N O O O O O N C N ZZnn11 Zn2 ZZnn11 Zn2+ ZZnn11 Zn2+ O Glu183 N C Glu183 C NH NH O w1 Wat1 O Glu183 O HN O O w2 Wat2 O O NH O O w1 (1) water-adding step H H H (2) ring-opening step H O H 1 H NH OH NH H w2 O N O O N HN N HN 1 H NH 2 5 C O(H) C C 3 4 N His178 Glu122 O(H) O(H) N Glu122 N Glu122 His178 HN His178 HN HN 77 ES TI EP 78 79 80 The crystal structures show different coordination geometries of M1 for each metal 81 type, while M2, usually a zinc ion, shares a common tetrahedral coordination. In native 82 creatininase (Zn-Zn enzyme), Zn1 has a distorted tetrahedral geometry bound to three protein 83 ligands, namely, Glu34, Asp45, and His120, a carbonyl oxygen of creatinine substrate and a 84 water molecule (Wat1). Zn2 has a well-ordered tetrahedral geometry bound to three protein 85 ligands, namely, His36, Asp45, and Glu183 and Wat1. The first metal Zn1 can be replaced 86 with other divalent cations (e.g., Mn2+, Mg2+or Co2+), providing highly active forms of the 87 enzyme 13. In a Mn-activated enzyme (Mn-Zn enzyme), Mn1 has a square-pyramidal 88 geometry bound to the three protein ligands, the substrate and two water molecules (Wat1 89 and Wat2). Currently, a total of thirteen crystal structures of the enzyme are deposited in the 90 Protein Data Bank (PDB), including the native enzyme 9, 10 and the enzyme-inhibitor/product 91 complex 11, 12, but none of them have a substrate bound. The lack of structural information for 92 the Michaelis complex could lead to an incomplete understanding of the catalytic mechanism 93 of creatininase. 94 On the basis of the available X-ray structures 10-12, it is thought that the enzyme 95 degrades creatinine via a two-step mechanism, similar to the urease-related amidohydrolase 96 enzymes, such as urease 14, phosphotriesterase 15, dihydroorotase 3, 16 and of the 97 aminopeptidases 17. Specifically, these metalloenzymes initiate the addition of the 98 nucleophilic water molecule that is located between the two metal ions (Wat1) followed by a 99 ring-opening step assisted by a conserved aspartate/glutamate residue that functions as a 100 catalytic base/acid 2. In creatininase, the first step is likely to follow the same reaction 101 mechanism, but in the second step two possible ring-opening pathways have been proposed 102 for protonating the leaving amide group and concomitant carbon-nitrogen bond cleavage (see 103 Scheme 2). They differ in the nature of the proton donor: either a protein residue (His178) 10 104 or a neighboring water molecule (Wat2) 11, 12 that is located between Glu122 and Zn1 could 105 play this role. His178 is located in a mobile flap (residues 168–180) that can be in an open or 106 closed conformation, while Glu122 is located at the M1 site. His178 and Glu122, as well 107 as other residues (Ser78, Tyr121, Trp154, Trp174) around the active site pocket (see Figure 108 S1), also influence the creatininase activities 12.
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