Mechanisms of ammonia activation and ammonium ion inhibition of quinoprotein methanol dehydrogenase: A computational approach

Abstract

The mechanism of methanol oxidation by quinoprotein methanol dehydrogenase (MDH·PQQ) in combination with methanol (MDH·PQQ·methanol) involves Glu-171 general base removal of the hydroxyl proton of methanol in concert with hydride equivalent transfer to the quinone carbon of pyrroloquinoline quinone (PQQ) and rearrangement to hydroquinone (PQQH₂) with release of formaldehyde. Molecular dynamics (MD) studies of the structures of MDH·PQQ·methanol in the presence of activator NH₃ and inhibitor have been carried out. In the MD structure of MDH·PQQ·methanol·NH₃, the hydrated NH₃ resides at a distance of ≈24 Å away from methanol and the ortho-quinone portion of PQQ. As such, influence of NH₃ on the oxidation reaction is not probable. We find that competes with the substrate by hydrogen-bonding to Glu-171 such that the complex is not reactive. Ammonia readily forms imines with quinone. Imines are present in solution as neutral () and protonated () species. MD simulations establish that the derivative of structure is unreactive because of the nonproductive means of methanol binding. The structure obtained by the MD simulations with the neutral imine of MDH·PQQ(NH)·methanol structure is similar to the reactive MDH·PQQ·methanol complex. This active site geometry allows for catalysis of hydride equivalent transfer to the of PQQ(NH) by concerted Glu-171 general-base removal of the HOCH₃ proton and Arg-324H⁺ general-acid proton transfer to the imine nitrogen. Enzyme-bound derivative of PQQ [PQQ(NH)] and CH₂O product are formed.

Methanol dehydrogenase (MDH) oxidoreductase is a soluble periplasmic quinoprotein important in oxidizing methanol to formaldehyde during growth of bacteria on methane (1–5). Pyrroloquinoline quinone (PQQ) or methoxatin (old name) is the noncovalently bound prosthetic group of MDH (Fig. 1a). The enzyme catalyzes methanol oxidation with PQQ reduction to hydroquinone (PQQH₂) and release of formaldehyde. This reaction is followed by two sequential single-electron transfers to the cytochrome c_L, during which PQQH₂ is oxidized back to the quinone. In vitro, the oxidation of methanol occurs at pH 9 with phenazine methosulphate as an artificial electron acceptor. The rate-limiting step is the oxidation of substrate. Ammonia and methylamine are activators (6–8), but ammonium ion and high concentrations of ammonia act as inhibitors (8–10). Ortho-quinones react easily with primary amines to form carbinolamines, which are in equilibrium with the corresponding imines (11). Imines readily undergo nucleophilic addition by specific and general acid catalysis.

Fig. 1.

Structure of the PQQ (a), modified PQQ, (b), and PQQ(NH) (c).

Harris and Davidson (9) reported that a very small but certain spectral perturbation occurs on addition of NH₃ to an aqueous solution of PQQ, which has been associated with the formation of an iminoquinone adduct. This spectral feature was not seen on addition of NH₃ to a solution of MDH·PQQ (8). Thus, it was concluded that the PQQ moiety of MDH·PQQ does not react with NH₃ at low concentrations to form an imine (9). It has occurred to us that there are alternate explanations for the latter observation.

Molecular dynamics (MD) simulations provide proper alignment of the active site residues in the enzyme-substrate and enzyme-intermediate complexes. Comparison of the x-ray coordinates of MDH intermediate formed during methanol oxidation and the ab initio structure of hydride adduct to the C5 of PQQ (12) established a mechanism (3), which involves the hydride transfer from methanol to PQQ to provide PQQH₂ (Fig. 2). This mechanism has been elaborated by MD simulation studies of MDH (13–15) and soluble glucose dehydrogenase (sGDH) (16) reactions. Further, the x-ray structure of sGDH·PQQ·glucose is best explained by hydride transfer in the oxidation of glucose by sGDH (17). In the present study, we examined the obvious means by which can influence the rate of oxidation of methanol by probing the active site of MD structures of MDH·PQQ·methanol and the intermediate complex MDH·PQQH in the presence of NH₃ and also . In addition, MD structures of the and imine derivatives of MDH·PQQ·methanol have been investigated.

Fig. 2.

Hydride transfer mechanism (from MD studies) of methanol oxidation by the MDH.

Methods

The partial atomic charges of PQQ, PQQ(NH), and (Fig. 1) were obtained by means of quantum chemical calculations by using gaussian 98 (18). In this procedure, Ca²⁺ ion was included at an appropriate coordinating distance to the N6, O7A, and O5/N5 of the coenzyme. PQQ(NH) and were optimized at the HF/6–31+G(d,p) level, and the electrostatic potential was calculated at the MP2/6–31+G(d,p) level by using the Merz–Singh–Kollman scheme (19). The restrained electrostatic potential method was used to fit the electrostatic potential by using an atom-centered point charge model (20).

From the x-ray structure (Protein Data Bank ID code 1G72) of the enzyme-bound intermediate (12), the MDH·PQQ is modeled so that the C5 of PQQ is a planar, rather than a tetrahedral, configuration of the crystal structure. Subsequently, methanol was docked into the active site of that complex (13) by using the program sybyl (Version 6.3; Tripos, St. Louis) to provide the MDH·PQQ·methanol structure. Such a structure was used in MD simulations with or modified PQQ. The is placed in the active site, proximal to Glu-171, such that a hydrogen bond occurs between them. The O5 oxygen at the C5 of PQQ was replaced appropriately by NH and in MDH·PQQ·methanol to provide MDH·PQQ(NH)·methanol and structures, respectively. Each structure was minimized for 500 steps of steepest descent and 1,500 steps of adopted basis Newton-Raphson methods by using the program charmm (21) (version c27b4) and charmm27 all-atom force field parameters (22). Ammonia parameters reported by the MacKerell group (23) were used. The system was solvated in an equilibrated TIP3P (24) water sphere of 42-Å radius by using the center of mass of PQQ or modified PQQ bound to heavy subunit A as the origin. Any solvent molecule within 2.8 Å of heavy atoms was deleted. The modeled structures contain a total of ≈37,000 atoms.

Adopting similar modeling and the MD procedures of MDH·PQQ·methanol structure (13), stochastic boundary molecular dynamics was carried out on the four enzyme-reactant structures (MDH·PQQ·methanol·NH₃, , , and MDH·PQQ(NH)·methanol) by using the program charmm (21). An integration time step of 1.5 fs was used, and nonbonded interactions were truncated at a distance of 12 Å. shake was applied to all covalent bonds involving hydrogen atoms (25). Similar MD procedures were followed for the intermediate structures MDH·PQQH·NH₃ and . As the active sites in both the heavy subunits of MDH were identical, the analyses were confined to the trajectories of the first heavy subunit. MD structures were averaged during the period 2.025–4.00 ns for analyses.

Results and Discussion

MDH·PQQ·Methanol. Interactions of the active site residues and Ca²⁺ coordination pattern of the reactant structures are given in Figs. 3 and 4, respectively. Our earlier MD studies had suggested that the hydride transfer mechanism of methanol oxidation by MDH (Fig. 2) involves Glu-171 general-base abstraction of the methanol hydroxyl proton concerted with hydride transfer from the methanol methyl carbon to the C5 of PQQ (13, 15). The reaction is assisted by hydrogen bonds of Arg-324 to the PQQ carbonyl O5 and O4 oxygens, and crystal water Wat1 to the O5 of PQQ (Fig. 3a).

Fig. 3.

Active site residues in the MD-averaged structures: MDH·PQQ·methanol (a), MDH·PQQ·methanol·NH₃ (b), (c), (d), and MDH·PQQ(NH)·methanol (e). Ca²⁺ is omitted for clarity. Nonbonded interactions are shown by dashed lines. Distances between the heavy atoms are in Å.

Fig. 4.

Ca²⁺ coordination in the MD-averaged structures: MDH·PQQ·methanol (a), MDH·PQQ·methanol·NH₃ (b), (c), (d), and MDH·PQQ(NH)·methanol (e). Distances are in Å.

MDH·PQQ·Methanol·NH₃. Initially, NH₃ is close to Glu-171 but moves away and resides ≈24 Å from the active site after 500 ps of dynamics (Fig. 3b). Such a feature can be interpreted as the diffusion of NH₃ away from the active site to interact with the waters on the surface and per se has no influence on the reaction mechanism. A few structural changes are noticed such as the absence of hydrogen-bond interactions involving Asn-255 (Fig. 3b), and the coordination of seven ligands to Ca²⁺ (Fig. 4b) instead of six Ca²⁺ ligands of the MDH·PQQ·methanol structure (Fig. 4a). The additional ligand to Ca²⁺ in the MDH·PQQ·methanol·NH₃ structure is the Asp-297 oxygen.

. MDH has a high affinity for methanol (K_m is 5–20 μM) but not for larger primary or secondary alcohols (1). This finding indicates that the active site is not large enough to accommodate a larger substrate (26). MD simulations with inclusion of show varied perturbations in the environment of the active site (Fig. 3c). The electrostatic environment in the active site allows to form hydrogen bonds with the quinone carbonyl oxygen O5 of PQQ (2.68 ± 0.10 Å), methanol oxygen (2.77 ± 0.09 Å), Glu-171 (2.61 ± 0.07 Å), and Asp-297 (2.57 ± 0.06 Å). Kinetic studies (10) have suggested that a salt bridge between and Glu-171 is likely to enhance the function of Ca² by withdrawing the negative charge from Glu-171 coordinated to Ca²⁺. This feature is in agreement with our MD results. The six-coordination pattern of Ca²⁺ in the complex (Fig. 4c) is similar to that in the MDH·PQQ·methanol structure (Fig. 4a).

MD simulations establish that the presence of at the active site prevents proper binding of methanol. Thus, the experimental effect of as an inhibitor (10) is observable in the MD structures. Methanol is seen to interact with Cys-104 and Trp-531 in the active site, such that it is not in position to react with PQQ or Glu-171.

Intermediate Complexes in the 2e^– Reduction of PQQ ( and MDH·PQQH·NH₃). The channel for diffusion of water molecules into the active site has been traced by MD studies on the MDH·PQQH structure (Fig. 2), and the possibility that promoter catalyst NH₃ or inhibitor can enter the active site in the same manner has been considered (13). Also, the pH optimum for enzyme activity is 9, such that the active site residues Glu-171 and Asp-297 of MDH both should be carboxylate entities. An active site comparison of the structure with MDH·PQQH is shown in Fig. 5. The guanido nitrogens of Arg-324 make bidentate hydrogen-bond interactions with the O5 (2.86 ± 0.11 and 3.15 ± 0.21 Å) and O4 (3.34 ± 0.23 and 3.12 ± 0.24 Å) oxygens of PQQH (Fig. 5b). Asp-297 forms a hydrogen bond with the HO5 of PQQH (3.45 ± 0.21 Å) but is not in position to assist migration of the H5 for the reaction PQQH → PQQH₂ (Fig. 2). Glu-171 is 3.35 ± 0.23 Å from the H5 of PQQH, and is 3.42 ± 0.29 Å from the H5. Interestingly, Wat1 oxygen is proximal (3.03 ± 0.30 Å) to the H5 of C5 during the entire simulation and very likely assists in the transfer of a proton to form PQQH₂. This feature, having water (Wat97/TIP3P) mediated by Glu-171 acting to assist the hydrogen transfer, also is observed in the MDH·PQQH structure (Fig. 2) (13, 15).

Fig. 5.

Active site residues in the MD-averaged intermediate structures: MDH·PQQH (a) and (b). Ca²⁺ is omitted for clarity. Nonbonded interactions are shown by dashed lines. Distances between the heavy atoms and those involving hydrogen atoms (in brackets) are in Å.

The orients in the active site with hydrogen-bond contacts to Glu-171 (2.61 ± 0.07 Å), Å), Wat1 (2.72 ± 0.08 and Wat97 (2.76 ± 0.10 Å) oxygens (Fig. 5b). In the structure, the coordination of Ca²⁺ includes the O7A (2.16 ± 0.05 Å) and N6 (2.35 ± 0.07 Å) of PQQ, Glu-171 carboxylate oxygens (2.26 ± 0.08 Å, 2.20 ± 0.07 Å), Asn-255 amide oxygen (2.27 ± 0.08 Å), Asp-297 carboxylate oxygen (OD2) (2.15 ± 0.05 Å), and Wat91 oxygen (2.38 ± 0.10 Å). A similar seven-coordination pattern of Ca²⁺ is noticed in the MDH·PQQH (15) structure but with coordination to Wat1 oxygen instead of Wat91 (observed in the structure). Also, an altered coordination of Asp-297 is noticed in the structure. The simulations on the MDH·PQQH·NH₃ structure show a tendency for NH₃ to be distant from the active site, and, henceforth, NH₃ would not play a role in the conversion of PQQH → PQQH₂.

Possible Involvement of and Imine Derivatives of PQQ. Before the discovery by the Klinman group (27) that the novel covalently linked ortho-quinone topaquinone serves as the coenzyme for amine oxidations in copper containing mammalian plasma amine oxidase, it was assumed that PQQ might be the cofactor. At that time, the mechanism for amine oxidation by PQQ was of interest in this laboratory (28–33) and elsewhere (34). A detailed study by Rodriguez and Bruice (33) established the formation of C5 imine of PQQ as an intermediate in amine oxidation. This finding leads to the expectation that NH₃ or MeNH₂ activation of the enzymatic alcohol oxidations also involve the C5 imine of PQQ. It is well known that addition reactions to centers are catalyzed by imine formation (33). The sequence of reactions in Eq. 1 can be considered for the amine activation of MDH.

The possible involvement of the intermediate has been explored by the MD simulation of structure. During dynamics of structure, the methanol moves far away (≈19 Å) from the active site toward the periphery of the water sphere (Fig. 3d). Thus, the mechanism given by Eq. 1 is very unlikely. The Ca²⁺ coordination pattern is seven, compared with six of MDH·PQQ·methanol structure (Fig. 4 a and d). Also, some active site interactions are disturbed.

For the oxidant to be the imine species , protonation of nitrogen must be concerted with hydride transfer. This process may take place with Glu-171 acting as general-base catalyst and Arg-324 acting as general-acid catalyst (Fig. 3e and Eq. 2).

Although the pK_a for proton dissociation from the positively charged guanido substituent of Arg-324 is ≈12, HN⁺ of Arg-324 would be suitable as a general-acid catalyst for the reaction because the pK_a for proton dissociation from should be ≈40. (Studies with the derivative of PQQ follow.)

The time variation plot of the MDH·PQQ(NH)·methanol structure shows that after 0.125 ns, the methanol oxygen is hydrogen-bonded (2.78 ± 0.16 Å) to the carboxylate oxygen (OE1) of Glu-171. This feature remains throughout the simulation (Fig. 6a). The stereo plot of the active site residues of the MDH·PQQ(NH)·methanol structure is shown in Fig. 7. Also, during most of the simulation, the methanol carbon approaches the electrophile C5 imine carbon of PQQ(NH) (3.83 ± 0.33 Å) sufficiently for hydride transfer (Fig. 6b). Arg-324 can assist the reduction of the PQQ(NH) because of the hydrogen bond between the imine nitrogen of PQQ(NH) and the guanido nitrogen (NH1HH11) of Arg-324 (3.05 ± 0.13 Å). Also, the N5 of PQQ(NH) is hydrogen-bonded to Asp-297 (3.04 ± 0.17 Å) and Wat1 oxygens (3.00 ± 0.18 Å) (Figs. 3e and 7). The carbonyl oxygen O4 at the C4 of PQQ(NH) is hydrogen-bonded to the amide nitrogen of Asn-387 (3.60 ± 0.36 Å) and forms bidentate interactions with the guanido nitrogens of Arg-324 (3.25 ± 0.23 Å and 2.87 ± 0.19 Å). As previously mentioned, these interactions are rather similar to those observed in the MDH·PQQ·methanol structure (Fig. 3a). An important difference is the absence of hydrogen-bonding between Asp-297 and the guanido nitrogen of Arg-324 in the MDH·PQQ(NH)·methanol structure.

Fig. 6.

Time-dependent variation of distances between the methanol oxygen and carboxylate oxygen (OE1) of Glu-171 (a), C5 quinone carbonyl carbon of PQQ and methanol carbon (b), methanol oxygen and carboxylate oxygen (OD1) of Asp-297 (c), and C5 carbon of PQQ and methanol oxygen of MDH·PQQ(NH)·methanol (d) structures. The dotted line corresponds to the separation of methanol hydroxyl hydrogen and OE1 of Glu-171 (a) and OD1 of Asp-297 (c).

Fig. 7.

Stereoview of the active site depicting important interactions of the MD-averaged (2.025–4.0 ns) MDH·PQQ(NH)·methanol structure. The methanol carbon is colored cyan. The Ca²⁺ coordination is in green, and coordinating distances are omitted for clarity and given in Fig. 4e. Nonbonded interactions are shown as red dashed lines. Average distances between heavy atoms are given in Å.

The methanol oxygen is 4.63 ± 0.21 Å from Ca²⁺ and is not close (5.00 ± 0.29 Å) to the carboxylate oxygen (OD1) of Asp-297 (Fig. 6c). During the entire simulation, Wat1 forms strong hydrogen bonds with both carboxylate oxygens of Asp-297 (2.63 ± 0.09 Å and 2.83 ± 0.12 Å), amide oxygen of Asn-255 (3.27 ± 0.20 Å), and imine nitrogen N5 of PQQ(NH) (2.99 ± 0.18 Å) (Fig. 3e). Another crystal water in the active site, Wat97 forms a hydrogen bond with the carboxylate oxygen (OE1) of Glu-171 (3.19 ± 0.51 Å). The Ca²⁺ coordination of the MDH·PQQ(NH)·methanol structure is seven (Fig. 4e), compared with the hexa coordination of Ca²⁺ in the MDH·PQQ·methanol structure (Fig. 4a).

Conclusions

It has been reported that the addition of NH₃ assists the oxidation of methanol to formaldehyde by the MDH·PQQ

complex, whereas inhibits this oxidation (8, 9). The object of this investigation has been to gain an understanding of these features. The MDH·PQQ·methanol structure exhibits little change on an addition of a single NH₃ and the formation of MDH·PQQ·methanol·NH₃ complex. Such a feature is caused by the NH₃ residing at ≈24 Å from the substrate and quinone moiety. Interestingly, MD studies show that takes the binding position of methanol, which in turn binds elsewhere. Thus, inhibits the enzyme catalysis.

If the enzyme-bound PQQ reacts with NH₃ (9) to form an imine, that imine can be present in the form of and/or . Based on early studies of nucleophilic addition to imines (11), it is expected that the PQQ () would be the optimal catalytic intermediate (Eq. 1). However, MD simulation studies of the structure established that the substrate resides 19 Å away from the oxidant. Therefore, the mechanism involving a intermediate (Eq. 1) is not possible. The MD structure of the MDH·PQQ(NH)·methanol is much the same as MDH·PQQ·methanol complex. The concerted general-base and general-acid catalysis of Eq. 2 is proposed. Glu-171 general-base removal of the HOC(H₂)H proton is concerted with Arg-324H⁺ general-acid assistance to the hydride transfer from methanol to PQQ(NH) to provide . General-base catalysis by Glu-171 and hydrogen-bonding of to Arg-324H⁺ also is involved in the oxidation of methanol in the absence of ammonia (13, 15).

Conversion of the enzyme-bound direct intermediate of hydride equivalent reduction of PQQ imine () to the 2e^– reduced imino hydroquinone (Eq. 3, step 3) may be assisted by water positioned in the structure. Such a positioning has been observed in the absence of ammonia in the conversion of the derivative of PQQ to PQQH₂. In all of the MD structures, Ca²⁺ coordination to the carbonyl O5 of PQQ or imine N5 of or protonated O5 of PQQH is absent. However, Wat1/Wat91 oxygen that coordinates to Ca²⁺ forms hydrogen bonds either to the O5 or N5 atoms and may indirectly contribute to the C5 polarization.