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. 2025 Jun 3;129(23):5664–5673. doi: 10.1021/acs.jpcb.5c00829

Protonation State of Active-Site Histidines, Reaction Mechanism and Stereoselectivity in β‑Alanine Synthase: A Computational Study

Wijitra Meelua 1,2, Ulf Ryde 3, Jitrayut Jitonnom 2,*
PMCID: PMC12169670  PMID: 40460037

Abstract

β-Alanine synthase (βAS), which is a dizinc metalloenzyme, catalyzes the irreversible hydrolysis of N-carbamyl-β-alanine (NCβA) to β-alanine. This enzyme has potential applications for β-amino acid production. Understanding the reaction mechanism and selectivity of βAS at atomic details can help design and engineer the enzyme for cascade biocatalysis. Here, the protonation states of two conserved active-site histidine residues (His262 and His397 in Saccharomyces kluyveri) of βAS were investigated by means of combined quantum mechanical and molecular mechanical (QM/MM) molecular dynamics (MD) simulation, as well as the ONIOM QM/QM′ approach. The calculations predicted that both His262 and His397 should be neutral for efficient catalysis. Furthermore, the βAS reaction mechanism and its stereospecificity toward a series of NCβA substrates containing different β2 and β3-β-alanine substitutions were studied, which suggested factors governing the origin of stereoselectivity of this enzyme. The mechanism for the conversion of NCβA into β-alanine, carbon dioxide, and ammonia by βAS involved four reaction steps: nucleophilic attack by a hydroxide ion, substrate protonation and formation of a zwitterionic intermediate, and C–N bond cleavage to produce β-alanine and carbamate, which is finally decomposed into carbon dioxide and ammonia. The rate-limiting step is the protonation of the amide nitrogen of the substrate by Glu159, with the overall reaction barrier (16.5 kcal/mol) consistent with the experimental data. In silico alanine scanning analysis of the reaction mechanism for four variants (His262Ala, His397Ala, Asn309Ala, and Arg322Ala) is performed, showing increased activation energies compared to the wild-type enzyme, which confirms the roles of these residues in catalysis. The results explain the enzyme’s preference for linear N-carbamyl substrates, as large and branched substrates cannot fit in the active site, restricted by the residue of the loop/region of the enzyme. Overall, we have demonstrated that a combined use of QM/MM MD and ONIOM models can be a promising strategy to elucidate possible protonation states of the ionizable residues in the enzyme active site prior to catalysis.

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1. Introduction

Dihydropyrimidine dehydrogenase (DHD, EC 1.3.1.2), dihydropyrimidinase (DHP, EC 3.5.2.2, also known as hydantoinase), and β-alanine synthase (βAS, EC 3.5.1.6, also called β-ureidopropionase, N-carbamyl-β-alanine amidohydrolase or N-carbamoylase) constitute the three enzymes involved in the reductive pyrimidine catabolic pathway (Scheme ). These enzymes, found across bacteria, yeast, animals, and plants, play critical roles in degrading uracil and thymine to β-alanine, ammonia, and carbon dioxide. − This pathway not only maintains cellular pyrimidine balance but also supplies β-alanine, a key metabolic intermediate. Deficiencies in these enzymes can result in severe toxicity in patients undergoing 5-fluorouracil treatment and have also drawn attention as potential drug targets and in stress response pathways in plants such as rice.

1. Reductive Pyrimidine Catabolic Pathway, in Which Three Important Enzymes Participate, Dihydropyrimidine Dehydrogenase (DHD), Dihydropyrimidinase (DHP), and β-Alanine Synthase (βAS).

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Beyond their biological significance, DHP/βAS enzymes are used in the industrial synthesis of β-amino acids, which are valuable building blocks in fine chemical and pharmaceutical production. − Their enzymatic selectivity can be tuned based on substituents at specific positions (e.g., R2 and R3 in Scheme ), enabling enantioselective synthesis. For example, Martínez-Gómez et al. demonstrated that 5- and 6-monosubstituted dihydrouracils, used as substrates with Sinorhizobium meliloti DHP (SmDHP) and Agrobacterium tumefaciens βAS (AtβAS), yield enantiopure β-alanine derivatives (e.e. >90% for R2 = Me; >95% for R2 = Phe). However, chemical synthesis remains inefficient and costly, and the molecular basis for the enzymes’ selectivity and catalytic differences is not fully understood. While SmDHP and AtβAS share similar active-site architectures, their differing substrate preferences are still unexplained. Recently, we rationalized SmDHP’s enantiopreference for substituted dihydrouracils, but the catalytic mechanism and selectivity of AtβAS remain poorly understood.

Crystal structures of βAS from Saccharomyces kluyveri (SkβAS) have recently been reported for both the apo and the NCβA-bound forms, at 2.0 Å resolution. The fold of the homodimer of SkβAS identifies it as a member of the AcyI/M20 family of metallopeptidases, , which consist of one or two domains with an α/β-fold. The SkβAS structure consists of two domains, a larger catalytic domain that contains a binuclear zinc center in the active site, and a smaller domain that mediates the majority of the intersubunit contacts (Figure a). At the active site of βAS, two zinc ions (Zn1 and Zn2) are bridged by an aspartate residue (Asp125) and a hydroxide ion (labeled as Wat1). Zn1 is coordinated to an aspartate (Asp125) and two histidine residues (His226 and His114), as well as a water molecule (Wat2) (Figure b, c). Zn2 is coordinated by Asp125, Glu160, His421, and the substrate. Glu159 serves as the catalytic base that activates the zinc-bridging hydroxide ion for the hydrolysis of NCβA. Six SkβAS structures have been solved so far (PDB entries 1R3N, 1R43, 2V8V, 2V8D, 2V8H, 2V8G), with the latter two capturing substrate or product bound at the active site.

1.

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(a) X-ray crystal structures of the SkβAS-E159A mutant complexed with the NCβA substrate (coordinates from PDB entry 2V8H, chain A). A dashed circle in panel a indicates the location of NCβA in the active site of SkβAS. (b, c) 3D and 1D schematic representations showing our ONIOM (QM:QM′) model system. High and low layers of the ONIOM model are denoted as A and B, respectively. Residues in the high layer are shown in ball-and-stick models in panel b and colored in blue in panel c.

The catalytic mechanism of SkβAS and the roles of its active-site residues have been previously elucidated through structural and mutagenesis studies. , Lundgren et al. proposed a mechanism for the βAS-catalyzed hydrolysis of N-carbamyl-β-alanine (NCβA) to β-alanine and carbamate, based on crystal structures (Scheme ). They suggested that βAS operates similarly to dizinc-dependent exopeptidases, where Glu159 acts as a proton shuttle by accepting a proton from the nucleophilic water and then transferring it to the β-amino group to facilitate breakdown of the tetrahedral intermediate. Mutational analysis of substrate-binding residues supports catalytic roles of Glu159, Asn309, and Arg322, while His262 and His397 are functionally relevant but not essential. Until now, the roles of His262 and His397 remain unclear. His262 was initially suggested to anchor the substrate’s carboxyl group, yet studies on the homolog AcyI show that mutation of the corresponding His206 drastically reduces catalytic efficiency, suggesting a catalytic role. , In SkβAS, His262 mutation causes only a modest activity loss, likely compensated by Gln229 and the dizinc center. His397’s role is even less clear; although previously linked to transition-state stabilization, structural data show it is too distant from the substrate, yet its mutation has a stronger effect than that of His262. These uncertainties highlight a key knowledge gap in the roles of His262 and His397 in catalysis and structural organization, supporting SkβAS as an important system for further investigation.

2. Proposed Mechanism for the βAS-Catalyzed Irreversible Hydrolysis of N-Carbamyl-β-alanine to β-Alanine and Carbamate, Which Is Hydrolyzed Further into Carbon Dioxide and Ammonia, As Suggested Experimentally by Lundgren et al.

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Understanding enzymatic mechanisms requires accurate knowledge of active-site protonation states, which influence catalysis, conformational dynamics, and structural stability. , Although X-ray crystallography reveals atomic positions, it often misses proton locations, especially on ionizable residues like histidine, which can adopt multiple protonation states. Computational tools such as empirical pK a predictors (e.g., PROPKA), quantum mechanical (QM)-based approaches (e.g., QM cluster, ONIOM), , machine learning, molecular dynamics (MD), and hybrid quantum mechanical/molecular mechanical MD (QM/MM MD) help assign these protonation states. For example, Uranga et al. used short MD simulations to evaluate histidine protonation in various proteins, whereas Kitao et al. carried out a series of MD followed by QM and QM/MM methods to probe histidine states in (6–4) photolyase. We previously applied QM/MM MD to investigate protonation effects of key residues on catalysis in GH18 chitinase and dizinc creatininase. , However, the protonation states of active-site residues in SkβAS and their influence on catalysis and structural stability remain unexplored.

In this study, we investigate the histidine protonation states and catalytic reaction of this SkβAS enzyme through a combination of QM/MM MD and ONIOM QM/QM′ calculations. We first determine the preferred protonation states of His262 and His397 in SkβAS and then investigate the reaction mechanism and stereoselectivity toward NCβA substrates with different β-substituents. Large models of the βAS active site are built to describe the mechanism and selectivity of SkβAS with DFT calculations. In addition, our computational models could account for previous mutagenesis experiments for key active-site residues (His262Ala, His397Ala, Arg322Ala, and Asn309Ala). Overall, we provide a comprehensive understanding of catalysis in SkβAS, which can be used to facilitate the development of biocatalytic cascades.

2. Computational Methods

2.1. QM/MM MD Simulations

Preliminary PROPKA calculations , using PDB entry 2V8H as input suggested that His262 and His397 are neutral at pH 7, because their calculated pK a values were below 7. However, we acknowledge that crystallization conditions such as the pH of ∼ 8.75 used in the X-ray study of the E159A mutant (PDB entry 2VH8) can significantly influence residue protonation states. To explore the ionizable states of the two active-site histidines (His262 and His397), four QM/MM MD simulations of the enzyme–substrate (ES) complex containing different protonation states of His262 and His397, namely, e262e397, e262p397, p262e397, and p262p397 (where 262 and 397 represent the histidine residue numbers, and e and p represent the neutral (HIE) and protonated (HIP) forms of the two histidines) were modeled (Figure ; HID was excluded, according to visual inspection on PDB). The starting structure was based on the X-ray crystal structure of SkβAS complexed with the NCβA substrate bound in the active site (PDB entry 2V8H). SkβAS forms a homodimer in the crystal structure, so it has two active sites and two substrates. In this study, we focus on the active site of the first monomer, chain A. A zinc-bound OH ion (Wat1), which is missing from the PDB entry 2V8H, was added between the two zinc ions. A short cycle of steepest descent energy minimization was then performed to relax the model system. The model was then solvated by a pre-equilibrated sphere of TIP3P water molecules with a 25 Å radius centered at the substrate carbonyl carbon. Any water molecule found within a 2.8 Å radius of a heavy protein/substrate atom was deleted, followed by relaxation of the solvent water sphere.

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Four active-site snapshots of ES complexes in different protonation states at His262 and His397 as obtained from QM/MM MD simulations: e262e397 (a), e262p397 (b), p262e397 (c), and p262p397 (d). The bridging water is mostly a hydroxide ion (OH–) but becomes a water molecule (HOH) in the e262p397 system by accepting a proton from Glu159.

To reduce the computational cost, stochastic boundary conditions were applied to the solvated system. The system is governed by Newtonian dynamics on a classical potential in the reaction region (r < 22 Å). The atoms in the reservoir zone outside a 25 Å radius were deleted. Atoms in the buffer zone (22 Å < r < 25 Å) were simulated with Langevin dynamics with friction and random forces stemmed from the bulk solvent that were not explicitly included in the simulation. The final size of the system contains ∼ 7250 atoms, including 16 substrate atoms and 561 water molecules. The atoms in the MM region were governed by the CHARMM27 all-atom force field, which has recently been applied successfully also in another QM/MM studies. , The QM regions for each model system consist of 118–120 atoms, including the NCβA substrate, two active-site water molecules (a hydroxide ion, Wat1, and a water molecule, Wat2), and two Zn2+ ions, as well as the side chains of His114, Asp125, Glu159, Glu160, His226, His262, Arg322, His397, and His421, which were treated quantum mechanically using SCC-DFTB. Hydrogen link atoms were placed between Cα and Cβ of the His and Asp residues and between Cβ and Cγ of the Glu residues.

The trajectories were propagated with a time step of 1 fs, and the SHAKE algorithm was applied to keep the length of covalent bonds involving hydrogen atoms fixed. All QM/MM MD simulations were conducted in the NVT ensemble for 500 ps using the CHARMM suite of molecular simulation programs. ,

2.2. ONIOM QM/QM′ Calculations

The final snapshots obtained from the QM/MM MD simulations were chosen and divided into two layers, which were treated with different theory levels according to the ONIOM methodology. The ONIOM QM/QM′ calculations were set up in a similar manner as in our previous studies − and were performed on the SkβAS active site residues in a sphere of 5 Å around the carbonyl carbon atom of the substrate. The high-level layer has the same size as in the QM region of the QM/MM MD study, but it includes two additional polar residues (Gln229 and Asn309) to include steric and electrostatic interactions when the stereospecificity is studied.

The full ONIOM model has a total of 651 atoms for the e262e397 system, consisting of the high-level region (125 atoms; see layer A in Figure b,c) that includes the substrate, two zincs, the first coordination shell, and amino acid residues that are directly involved in the reaction (His114, His226, His262, Arg322, His397, His421, Asp125, Glu159, and Glu160) as well as Gln229 and Asn309. Atoms in the low-level layer (layer B) are shown in Figure c. Implicit hydrogen link atoms were used to complete the valence between the two layers.

Layer A was optimized with DFT using the B3LYP, M06-2X, B97D, ωB97XD, and MPWB1K functionals and the 6-31+G­(d,p) basis set (Figure S1 and Table S1). Among these, the B97D functional was selected for all subsequent calculations due to its reliable performance in reproducing experimental barriers and effectively accounting for dispersion interactions. The layer B was treated with the semiempirical PM7 method. This method (QM′) was chosen for mechanistic investigations since it has demonstrated very good results in the theoretical description of enzymatic processes, − especially the investigations of enzyme stereospecificity. The optimization procedure was carried out using the Gaussian software versions 09/16. The reaction mechanism was explored through relaxed scans along the reaction coordinates.

3. Results and Discussion

3.1. Protonation States of His262 and His397

Prior to elucidating the catalytic mechanism of SkβAS, it is essential to determine the protonation states of the two key histidine residues, His262 and His397. We performed QM/MM MD and ONIOM QM/QM′ calculations on four possible protonation state combinations (e262e397, e262p397, p262e397, and p262p397) to assess their influence on the active-site dynamics, structural stability and enzyme catalyzed reaction. Representative snapshots of the equilibrium ES structures for the four model systems are depicted in Figure , and their active-site geometries and flexibilities are given in Table S2 and Figures S3–S8. We found that in all systems, the substrate is maintained within the enzyme active site throughout the simulations, and the systems reached equilibrium after 200 ps, with the RMSD values of ∼0.2 Å (Figure S2). However, the four QM/MM MD trajectories are similar except the position of the water molecule and the bridging OH– ion with respect to Zn1, the substrate, and Glu159 (the Zn1–O­(H2O), CS–OOH, and HOH–O­(Glu159) distances, respectively; Figures S3 and S4). The e262e397 and p262e397 systems are found to maintain these geometries close to the X-ray crystal structure: the Zn1–O­(H2O), OOH–CS, and HOH–O­(Glu159) distances are 2.47, 2.53, and 2.10 Å for e262e397 and 2.48, 2.54, and 2.14 Å for p262e397 (Figures S5 and S6). The bridging water molecule primarily adopts the form of a hydroxide ion (OH–), highlighting its potential role as a nucleophile. However, in the e262p397 system, the water molecule (HOH) instead gains a proton from Glu159, suggesting a shift in its chemical behavior due to changes in the protonation states of the nearby histidines. The protonation of His397 strongly influences the water ligation at Zn1, with relatively large Zn1–O­(H2O) distances in e262p397 and p262p397 (4.64 and 2.91 Å, respectively; Table S2 and Figure b,d), compared to the deprotonated His397 systems (2.47 Å), which indicated that His397 must be deprotonated so that it could maintain Wat2 as the fifth ligand of Zn1.

The active site shows limited fluctuations in the metal-binding residues and the substrate’s carbamyl group, while His262, Asn309, and Arg322, which help anchor the NCβA carboxylate, are more flexible (Figure S7). Among the systems studied, e262e397 best matches the X-ray structure, maintaining His262’s orientation and a specific hydrogen bond with the carbamyl group (Figures and S8). The other protonation states disrupt this interaction, shifting His262 and altering hydrogen-bonding with the NCβA carboxylate. These results highlight how local protonation states shape the active site’s structure and dynamics. Overall, the QM/MM MD simulations show that e262e397 most accurately reproduces the ES geometry observed crystallographically.

We further examined which of the four protonation states yields the lowest potential energy surface (PES) using ONIOM QM/QM′ calculations. The ONIOM potential energy profiles for e262e397, p262e397, e262p397 and p262p397 were computed at the B97D/6-311+G­(2d,2p):PM7 level, as shown in Figures and S9. For comparison, all calculations were performed while maintaining the carbamyl-His262 interaction, as observed by crystallographic evidence. The evolution of key bond distances for each system is summarized in Table S3. Among the systems, e262e397 exhibited the most energetically favorable PES, with the lowest overall barrier of 16.5 kcal/mol at TS2 and an overall reaction exothermicity of –14.8 kcal/mol. In contrast, protonated states led to elevated barriers, especially when His397 was protonated: 21.4 kcal/mol at TS3 for p262e397, 20.9 kcal/mol at TS2 for e262p397, and 19.4 kcal/mol at TS2 for p262p397. These trends suggest that protonation of His262 or His397 increases the activation energy by destabilizing key intermediates and transition states. Mulliken charge and structural analyses support that histidine protonation perturbs enzyme reactivity by reducing electrostatic effects at the metal center, thereby raising the energy landscape and impairing carbamate decomposition (see Supporting Information for details). Thus, the neutral protonation states of His262 and His397 provide an optimal electrostatic environment for catalysis. The optimized structures and noncovalent interactions observed along the e262e397 pathway are illustrated in Figures – and S10. Based on these findings, we focus our mechanistic discussion of SkβAS-catalyzed hydrolysis of N-carbamyl-β-alanine on the neutral e262e397 system, which provides the most plausible and energetically favorable reaction mechanism.

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Calculated potential energy profiles for SkβAS-catalyzed hydrolysis of NCβA of the most favorable protonation state, e262e397, as obtained from the ONIOM calculations at the B97D/6-311+G­(2d,2p):PM7 level of theory. The PESs for all other pronation states can be found in Figure S9. An alternative pathway for carbamate decomposition is also indicated with red dashed lines.

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First step of the reaction: nucleophilic attack and formation of the gem-diol intermediate. Stationary points of the proposed reaction (ES, TS1, and IM1) are shown in 3D as sticks (top) and in 2D (bottom). Distances are in Å. Bond breaking/forming distances are indicated with red dashed lines.

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Fourth step of the reaction based on the preferable pathway of carbamate decomposition: Concomitant proton transfers to the carbamate nitrogen atom (Ncar) via the amino group of the β-alanine product. Stationary points of the steps (IM3, TS4, and EP) are shown in 3D as sticks (top) and in 2D (bottom). Distances are in Å. Bond breaking/forming distances are indicated with red dashed lines. Alternative pathway for the fourth step of the reaction is included in Figure S11 of Supporting Information.

3.2. Catalytic Mechanism

The ONIOM QM/QM’ calculations suggested four important steps for the degradation of NCβA into β-alanine product, carbon dioxide, and ammonia by SkβAS. This involved (i) nucleophilic attack by the bridging hydroxide ion and formation of the gem-diol intermediate, (ii) protonation of the amide nitrogen and formation of a zwitterion, (iii) deprotonation of gem-diol and the C–N bond cleavage to yield carbamate and β-alanine products, and finally (iv) carbamate decomposition to produce carbon dioxide and ammonia. Detailed mechanisms and relevant transition states, as well as structures of the intermediates are provided and discussed below (Figures –).

The first step is a nucleophilic attack of the bridging OH– ion on the CS atom of the CS–NS amide bond (Figure ), which leads to the formation of the gem-diol tetrahedral intermediate (ES → IM1). During this step, the Glu159 side chain forms two H-bonds with the NS atom of substrate and the hydroxide, which decrease by 0.2–0.4 Å from 2.18 and 1.78 Å at ES to 1.78 and 1.57 Å at IM1, respectively. At the same time, the OOH–CS distance shortens from 2.50 Å at ES to 1.85 Å at TS1 and 1.50 Å at IM1. The barrier of this step is 7.5 kcal/mol. A tetrahedral geometry of the substrate in IM1 is observed, as evident by elongation of the CS–NS (1.30 Å at ES to 1.48 Å at IM1) and the −CO bonds (1.28 Å at ES to 1.35 Å at IM1). In IM1, the hydroxide is covalently attached to the substrate and has more asymmetric Zn1–OOH/Zn2–OOH distances (2.03 and 2.52 Å at IM1, compared to ∼2.0 at ES). During this step, Zn2 plays a catalytic role by stabilizing the oxyanion of the tetrahedral intermediate, whereas His262 does not, as indicated by the elongation of the (His262)­NH–OS distances from 1.70 Å at ES to 1.82 Å at IM1. These findings support previous experimental evidence that His262 functions primarily in substrate anchoring rather than direct catalysis.

In the second step, the protonated Glu159 residue plays the role of an acid and delivers its proton to the NS atom of the β-amino acid moiety of the substrate (Figure ) to form a zwitterion intermediate. From IM1, the second transition state for the proton transfer (TS2) and the resulting zwitterion intermediate (IM2) have been optimized, as shown in Figure . At TS2, the O···H···NS distances between Glu159 and NS are 1.34 and 1.38 Å. During this step, the CS–OH bonds of the gem-diol moiety shorten by 0.1 Å, while the CS–NS distance is slightly elongated by 0.1 Å (1.48 Å at IM1 to 1.58 Å at IM2). The zwitterion is not stable by itself but is stabilized by hydrogen bonding with the negative charge side chain of Glu159, which is also stabilized by a second water located between Zn1 and His397. This is evident by the shortening of H-bond distances created by Glu159 to the gem-diol and the NS atom, as well as the decrease of a H­(H2O)–O­(Glu159) H-bond from 1.96 Å at TS2 to 1.84 Å at IM2. This emphasizes the role of water coordination in dizinc metalloenzyme, as previously reported. Again, the unchanged (His262)­NH–OS distances further support the conclusion that this residue does not play a catalytic role. TS2 has a barrier of 16.5 kcal/mol, making it the highest point on the energy profile for e262e397. Therefore, this step is the rate-determining step for the hydrolysis of NCβA into β-alanine. Previous experimental rate constants (k cat = 5.3–25.7 s–1  , ) have been measured for this reaction catalyzed by βAS, which corresponds to energy barriers of ca. 15–17 kcal/mol using the transition-state theory. The computed barrier of 16.5 kcal/mol is consistent with this experimental barrier, validating the current model for mechanistic study in this work.

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Second step of the reaction: protonation of the amide nitrogen and formation of the zwitterion intermediate. Stationary points of the proposed reaction (IM1, TS2, and IM2) are shown in 3D as sticks (top) and in 2D (bottom). Distances are in Å. Bond breaking/forming distances are indicated with red dashed lines.

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Third step of the reaction: deprotonation of the gen-diol and the collapse of the tetrahedral intermediate via CS–NS bond cleavage. Stationary points of the proposed reaction (IM2, TS3, and IM3) are shown in 3D as sticks (top) and in 2D (bottom). Distances are in Å. Bond breaking/forming distances are indicated with red dashed lines.

The third step is the Glu159-assisted CS–NS bond cleavage that leads to the generation of the carbamate and β-alanine products. At TS3, the proton transfer distances ((Glu159)­O···HOH···OOH) between the OH group of the substrate and the Glu159 side chain are 1.33 and 1.34 Å, respectively. The result of proton transfer yields β-alanine and carbamate, as evidenced by the lengthening of CS–NS distance from 1.58 Å at IM2 to 3.07 Å at IM3. A slight reduction in the (His262)­NH–OS distance (∼0.04 Å) at TS3 suggests that the imidazole side chain of His262 may stabilize the tetrahedral transition state during C–N bond cleavage. In IM3, the protonated Glu159 orients to stabilize β-alanine via the NH2-group. The barrier is calculated to be 15.5 kcal/mol relative to ES. IM3 is 5.0 kcal/mol above the ES energy, indicating that the N-carbamyl-β-alanine degradation is an endothermic process. Note that we did not consider the regeneration process after the CS–NS bond cleavage, which would involve the leaving of the products and the formation of a hydroxide ion, bridging the two zinc ions, originating from a water molecule.

The final step is the decomposition of carbamate into carbon dioxide and ammonia activated by Glu159. Here, two mechanistic scenarios have been tested in which the transfer of a Glu159 proton to carbamate occurs directly or proceeds via the amino group of the β-alanine product (Scheme ). Geometries of the two mechanistic pathways are depicted in Figures and S11. The calculated potential energy profiles in Figure clearly indicate that the mechanism involving concomitant proton transfers to a carbamate nitrogen atom (Ncar) via the amino group of the β-alanine product is energetically preferred, with a barrier of 12.3 kcal/mol relative to ES (21.2 kcal/mol for the alternative pathway via the direct proton transfer). The final products (CO2, NH3, and β-alanine) are largely stabilized through attractive and van der Waal interactions (see EP, Figure S10).

3. Two Mechanistic Scenarios for Carbamate Decomposition by SkβAS .

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a (a) Mechanism involving concomitant proton transfers to a carbamate nitrogen atom (Ncar) via the amino group of β-alanine product. (b) Alternative mechanism involving a direct proton transfer from Glu159 to the Ncar atom.

3.3. In Silico Alanine Mutations

Mutagenesis experiment on SkβAS has shown that Glu159 and Arg322 are crucial for catalysis, while His262 and His397 are functionally important but not essential. Asn309 is also important, as its alanine mutation significantly reduced the k cat value. To evaluate the catalytic effects of these active-site residues, we employed the ONIOM QM/QM′ model to study the impact of four variants (H262A, H397A, R322A, and N309A) on the enzyme kinetics (i.e., reaction barriers). The transition-state energies (TS2 and TS3) for the four mutated enzymes are given in Table . It was found that the energies of TS2 and TS3 were close in energy, but TS2 was the rate-determining step for all mutants except H262A. The calculated barriers for the rate-determining step of mutants H262A, R322A, and H397A are 23.4, 24.2, and 17.9 kcal/mol, respectively, which are all higher than the barrier in wild-type (16.5 kcal/mol). These results correlate quite well with experimental kinetic data for the mutants (Figure S12), although the increase in the barriers is typically overestimated (probably because the structures of the mutants were not equilibrated). These results illustrate the importance of these active sites. Arg322 shows the largest contribution and H262 is slightly more important than H397. Overall, these calculated barriers reproduce the experimental trend and provide a rationale for the observed activity difference in site-directed mutagenesis. In N309A, we further elucidate the steric effect of this polar residue on the kinetic rate, as detailed in Table . This mutant significantly impacts the kinetics, as evidenced by a calculated barrier reduction from 16.5 kcal/mol at TS2 in the wild type to 15.7 kcal/mol at TS2 in N309A, which aligns with experimental observations. Therefore, our ONIOM calculations successfully reproduce the reaction kinetics of the variants, supporting the reliability of the ONIOM model in investigating the SkβAS mechanism.

1. Values of Activation Energies at TS2 and TS3 during the Hydrolysis of NCβA by WT and Mutant SkβAS (H262A, H397A, and R322A).

Mutants TS2 TS3 Ea(expt)
WT 16.5 15.5 16.7
H262A 22.1 23.4 18.2
R322A 24.2 23.8 20.7
H397A 17.9 15.5 18.3
N309A 15.7 12.9  
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a

Values are in kcal/mol.

3.4. Enzyme Selectivity toward Substituted Substrates

The substrate pocket of SkβAS is composed of mostly charged residues and some hydrophobic residues, some of which have been shown to influence substrate selectivity. , To probe the selective properties of the enzyme pocket toward a series of substituted carbamyl substrates, we theoretically modeled the SkβAS reaction with five NCβA substrates (1)–(5) bearing different R groups at the positions R2 and R3, as shown in Table and Figure . The whole energy profile and noncovalent interactions through the reaction path were calculated for each studied substrate (Figures S13 and S14). It is clearly shown that all substrates were hydrolyzed by the enzyme through the same mechanistic pathway as for the unsubstituted substrate, and that step 2 (IM1 → TS2) is the rate-determining step. The values of computed barriers (E a) of this key step for each substrate (1)–(5) are included in Table . The E a values decrease in the following order: R2, R3 = Ph, H (4) > H, Me (5) > NH2, H (3) > H, H (1) and Me, H (2). This trend indicates that substrates (1) and (2) are the most reactive substrates. A comparison of E a values between substrates (2) and (5) clearly demonstrates the preference for R2 substitution. Furthermore, the highest barrier of 28 kcal/mol in (4) is obtained for the bulky phenol R2 substituent (Figure c). NCI analysis further reveals a varying degree of weak (stabilizing) interactions at the reactant state for each substrate, with S167, Y249, and G396 contributing to favorable binding at R2 position. These results support the preference of the enzyme for linear carbamyl substrates, as bulky substrates cannot fit in the SkβAS active-site pocket. Similar findings are also observed in a Rhizobium radiobacter N-carbamoyl-β-alanine amidohydrolase.

2. Activation Energies (E a) and Binding Energies (BE) Calculated for the Enzyme Complexed with Substrates Containing Different R Groups .

3.4.

Substrate R2 R3 Ea(expt.) Ea(Calc.)
1 H H 15.80 16.5
2 CH3 H 15.83 15.6
3 NH2 H 18.90 19.6
4 Phenyl H 18.99 28.9
5 H CH3 18.73 22.9
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a

Values are in kcal/mol.

8.

8

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Comparison of binding modes of the R2, R3-substituted NCβA substrates (a) (H, Me), (b) (Me, H), and (c) (Ph, H) with (d) the unsubstituted substrate (H, H). Substrates are highlighted as gray shades; yellow shades indicate the substituent groups, with surrounding residues (including steric clash) shown in yellow text.

4. Conclusions

Using the X-ray structure of SkβAS provided by Lundgren et al., combined with QM/MM MD simulations and ONIOM (QM/QM′) computations, we investigated the protonation state of two active-site histidines, His262 and His397, and their effect on the active site dynamics and enzyme kinetics. Four protonation tautomers of His262 and His397 were assessed, and the neutral configuration (e262e397) consistently provided the best agreement with the crystallographic structure and the lowest energy pathway for NCβA hydrolysis. Our QM/MM MD results showed that protonation of either histidine caused significant structural deviation from the X-ray structure, especially affecting His262. Additionally, ONIOM energy profiles revealed that protonated systems raised the activation barrier, especially for the carbamate decomposition step. Notably, His397 must remain deprotonated to preserve the coordination of Wat2 as the fifth ligand of Zn1. Charge and geometric analyses further confirmed that protonation perturbs the electrostatic environment at the metal center, weakens hydroxide binding, and reduces transition-state stabilization. Mechanistically, the second step involving protonation of the amide nitrogen and zwitterion formation was identified as the rate-determining step with a barrier of 16.5 kcal/mol. During reaction, His262 is unlikely to stabilize the oxyanion of tetrahedral intermediate, explaining previous experimental observations. The computed activation barriers for H262A, H397A, and R322A mutants were significantly higher than in the wild-type enzyme, consistent with experimental data. The reactivity of the substrates is as follows: R2, R3 = (H, H) = (Me, H) > (NH2, H) > (H, Me) > (Ph, H), indicating a preference for small substrates with a methyl substituent at the R2 position. Collectively, our results highlight that the neutral protonation states of His262 and His397 provide a favorable electrostatic environment for catalysis and preserve the structural integrity of the active site. This work underscores the utility of integrating QM/MM MD and ONIOM calculations to predict functionally relevant protonation states in metalloenzymes and offers mechanistic insights into SkβAS catalysis and substrate specificity.

Supplementary Material

jp5c00829_si_001.pdf (4.7MB, pdf)

Acknowledgments

We thank financial support from the University of Phayao and the Thailand Science Research and Innovation Fund (Fundamental Fund 2025, Grant No. 5026/2567) and the School of Science, University of Phayao (Grant No. PBTSC67019). Computer time and software are partly facilitated by the NSTDA Supercomputer Center (ThaiSC: Project ID pv812012). J.J. expresses sincere gratitude to Tanchanok Wanjai, Thamonwan Phedpha, and Bunchaporn Mongkolthong for their invaluable assistance with data collection and analysis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.5c00829.

  • ONIOM energy profiles for the SkβAS catalyzed hydrolysis of NCβA into carbamate and β-alanine products obtained with various DFT functionals (B3LYP, M06-2X, wB97XD, B97D, and MPWB1K); structural analysis of e262e397, e262p397, p262e397, and p262p397 from QM/MM MD simulations and ONIOM QM/QM′ calculations; Mulliken charge analysis at the metal center; NCI analysis for e262e397 pathway and varying substrates at ES; alternative pathway for the carbamate decomposition; linear relationship between the computed and experimental-derived activation energies; relative potential energies for the hydrolysis of the substituted NCβA substrates; Cartesian coordinates for stationary points on the PES curves of four different His262/His397 protonation states (PDF)

The authors declare no competing financial interest.

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