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REVIEW

Determination of enzyme mechanisms by molecular dynamics: Studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase

Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA

Reprint requests to: Thomas C. Bruice, Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA; e-mail: tcbruice{at}chem.ucsb.edu; fax: (805) 893-2229.

	Abstract

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH^– [–HC5(O^–) –C4( = O) –] and PQQH [–HC5(OH) –C4( = O) –] converts to the hydroquinone PQQH₂ [–C5(OH) = C4(OH) –]. The main focus is on MD structures of MDH • PQQ • methanol, MDH • PQQH^–, MDH • PQQH, sGDH • PQQ • glucose, sGDH • PQQH^– (glucolactone, and sGDH • PQQH. The reaction PQQ PQQH^– occurs with Glu 171–CO2^– and His 144–Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5 = O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297–COOH would act as a proton donor for the reaction PQQH^– PQQH, if formed by transfer of the proton from Glu 171–COOH to Asp 297–CO₂^– in MDH. For PQQH PQQH₂, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171–CO₂^– in MDH and by Wat89 in sGDH).

Keywords: molecular dynamics; PQQ; quinoproteins; methanol dehydrogenase; soluble glucose dehydrogenase; hydride transfer mechanism

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04673404.

	Introduction

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
Molecular dynamics (MD) simulation, a computational approach, has gained popularity in the last 15 yr as a tool to understand the structure and function of biological macromolecules (Berendsen and Hayward 2000; Daggett 2000; Kollman et al. 2000; Tuckerman and Martyna 2000; Hansson et al. 2002; Karplus and McCammon 2002; Garcia-Viloca et al. 2004). MD studies enhance the understanding of the role of the active-site residues that are of fundamental importance for a microscopic description of the catalytic mechanism, and also for the design of effective enzyme mimics. Motions of crystal or bulk water can be observed, and in that way one can discover the channel of water into the active site of the enzyme.

MD technique uses a force field (parameterized on a test set of molecules) to describe the atomic interactions of a system in space and time by evaluating the classical Newtonian equation of motion. The method involves direct incorporation of electrons and can be used for the study of large and complex systems, unlike quantum mechanical calculations. Enzyme cofactors and ligands or substrates often require additional parameterization. The high-resolution crystal structure of the enzyme–substrate or enzyme–inhibitor complex is used as the starting coordinates for exploration of the structure and enzyme mechanism. Appropriate MD procedures allow the generation of either enzyme–substrate or enzyme–intermediate complexes with hydrogens (ionizable functions may be set to be ionized or protonated) in explicit solvent. Explicit solvation mimics the experimental conditions (at an appropriate pH and ionic strength of biomolecules). A realistic treatment of the system boundaries is carried out either by periodic boundary conditions (an elegant procedure, but computationally expensive; Brooks et al. 1985) or by enclosing the system of interest in a sphere, with restraints and stochastic forces acting at the boundary. The latter procedure is called stochastic boundary molecular dynamics (SBMD; Brooks and Karplus 1989), which is useful in the study of large multimeric solvated systems while probing a specified region of interest. The potential of SBMD to elucidate otherwise inaccessible details about enzyme mechanisms is illustrated by recent studies on quinoprotein dehydrogenases from our group (Reddy and Bruice 2003, 2004; Reddy et al. 2003), which form the focus of the rest of this review.

Quinoproteins are a class of redox enzymes bearing orthoquinone cofactors that function as oxidants in respiration and energy transduction (Duine and Frank Jzn 1981; Duine 1991; Anthony 1996; Davidson 2001). Quinoproteins occur in the periplasm of Gram-negative bacteria and as amine oxidases in mammalian plasma. They relay electrons from the oxidation of substrates to 1e^– electron-accepting proteins such as cytochromes, cupredoxins (amicyanin or azurin), and ubiquinone. Quinoproteins use different cofactors either bound noncovalently (or freely dissociable species) like pyrroloquinoline quinone (PQQ; Salisbury et al. 1979) or derived from amino acids in the protein backbone of the enzyme, such as topaquinone (TPQ; Janes et al. 1990), lysine tyrosylquinone (LTQ; Wang et al. 1996), tryptophan tryptophylquinone (TTQ; McIntire et al. 1991), and cysteine tryptophylquinone (CTQ; Datta et al. 2000). The unique properties of quinoproteins have led to biotechnological applications such as biosensors, biotransformation of specific products, and bioremediation (Matsushita et al. 2002).

PQQ-containing dehydrogenases, the bacterial enzymes, constitute the largest quinoprotein subclass (Anthony and Ghosh 1998; Oubrie and Dijkstra 2000; Anthony 2001; Davidson 2001; Matsushita et al. 2002). The enzymes occur either in soluble or in membrane-associated forms. PQQ is synthesized exogenously and then associates with an apo-protein to form the active holoenzyme. In the active site of these enzymes, PQQ is complexed with a divalent metal ion. The PQQ enzymes are broadly classified as alcohol dehydrogenases (ADH) and glucose dehydrogenases (GDH). ADH, which catalyzes the oxidation of alcohols into the corresponding aldehydes, is classified into three types. Type I comprises methanol dehydrogenase (MDH) and ethanol dehydrogenase. Type II ADH is a soluble quinohemoprotein. Ethanol dehydrogenase and Type II ADH, isolated from different pseudomonad species, oxidize primary and secondary alcohols (Keitel et al. 2000; Chen et al. 2002; Oubrie et al. 2002). Type III ADH, a membrane-bound quinohemoprotein, is found only in acetic acid bacteria (Inoue et al. 1989; Matsushita et al. 1994). GDH enzymes (from Acinetobacter calcoaceticus) are of two types, either bound to membrane, mGDH, or in soluble form, sGDH. The two types of GDH do not have sequence homology (Cleton-Jansen et al. 1988), nor are they kinetically and immunologically similar (Matsushita et al. 1989). mGDH catalyzes the oxidation of aldose sugars (mainly monosaccharides) to the lactones, with the electron acceptor being ubiquinone in the membrane. sGDH has broader substrate specificity than mGDH.

Our focus is on the PQQ enzymes, methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (sGDH). In the course of reaction, the orthoquinone PQQ is reduced to hydroquinone (PQQH₂). MDH catalyzes the oxidation of methanol to formaldehyde, a key step in biological metabolism in methylotrophic bacteria that have potential importance to biotechnology (Anthony 1986). The enzyme has a high affinity only for primary alcohols, with a K_m value of ~5–20 µM (Keitel et al. 2000; Anthony 2001). MDH uses cytochrome c_L as an electron acceptor to reoxidize the hydroquinone PQQH₂ to orthoquinone PQQ in two separate single-electron transfers (Anthony 1992). MDH has also been isolated from autotrophic bacteria, whose electron acceptor is cytochrome c-551 in Paracoccus denitrificans (Anthony 1986; Harris and Davidson 1993a; Xia et al. 2003). The experimental studies of MDH are complicated by enzyme endogenous activity (i.e., the enzyme catalyzes the reduction of an electron acceptor in the absence of any added substrate, and further, if allowed to react in this manner, the enzyme becomes inactivated). This substrate-independent activity is suppressed by cyanide, which also protects the enzyme against inactivation (Duine and Frank 1980; Harris and Davidson 1993b).

A few available X-ray structures of MDH • PQQ complexes are 1.94 Å (1H4I [PDB] ) from Methylobacterium extorquens (Ghosh et al. 1995); 2.4 Å (4AAH [PDB] ) from Methylophilus W3A1 (Xia et al. 1996); 1.9 Å (1G72 [PDB] ) from Methylophilus methylotrophus (Zheng et al. 2001); and 2.5 Å (1LRW [PDB] ) from Paracoccus denitrificans (Xia et al. 2003). These enzyme structures from different bacterial sources have nearly the same active sites and are also similar at the sequence level. Attempts to crystallize the substrate methanol in the active site of MDH have not yet been successful. Although a crystal structure reported the presence of methanol (Xia et al. 1999), subsequent refinement of the structure showed no methanol in the active site (Zheng et al. 2001). MDH is a heterotetramer with two heavy and two light subunits with a total mass of 150 kDa. The catalytic chemistry is located in each heavy subunit of MDH with similar features of the active site. The light subunit of MDH has an extended structure and along with the heavy subunit forms a dimer. The subunits cannot be reversibly dissociated, and the function of the light subunit is unknown. The heavy subunit of MDH has a -propeller fold, which is characteristic of eight four-stranded antiparallel -sheets.

sGDH oxidizes a wide range of pentose and hexose sugars, mono- as well as disaccharides, to their corresponding lactones (Matsushita et al. 1989). The physiological electron acceptor for reoxidation of PQQH₂ to PQQ in sGDH is unknown. Cytochrome b₅₆₂ (Dokter et al. 1988), in addition to artificial electron acceptors such as N-methylphenazonium methyl sulfate (Olsthoorn and Duine 1996), serves well as an oxidant. sGDH is presently used in the accurate monitoring of blood glucose using diabetic control test strips (Ye et al. 1993; Yamazaki et al. 2000). sGDH is a homodimer (Dokter et al. 1986; Geiger and Gorisch 1986) of 100 kDa and basic in nature (pI = 9.5). The four sGDH X-ray structures isolated from A. calcoaceticus are 1.72 Å (1QBI [PDB] ) apo enzyme (Oubrie et al. 1999b); 1.5 Å (1CRU [PDB] ) sGDH bound to PQQ and methylhydrazine inhibitor (Oubrie et al. 1999a); and 2.2 Å (1C9U [PDB] ) sGDH • PQQ and 1.9 Å(1CQ1 [PDB] ) sGDH • PQQH₂ • glucose structures (Oubrie et al. 1999c). Each subunit of sGDH binds PQQ, glucose, and three Ca²⁺ ions, with one of the Ca²⁺ ions located in the active site of the enzyme. The other four Ca²⁺ ions of sGDH have been implicated to have a role in the stabilization of the two subunits (Olsthoorn et al. 1997; Olsthoorn and Duine 1998b) and are absent in MDH. However, the Ca²⁺ ion in the active site is conserved in MDH and sGDH. Each subunit of sGDH is described by the -propeller fold structure (Oubrie et al. 1999b), similar to MDH. But the fold in sGDH is made of six four-stranded antiparallel -sheets. Although sGDH and MDH have certain similarities in their active sites, the amino acid sequence and the three-dimensional structures of the two enzymes are quite different.

The characteristic -propellers have been reported in several proteins, with eightfold -propeller of MDH observed in nitrite reductase (with heme as the prosthetic group; Fülöp et al. 1995), and PQQ enzymes such as ethanol dehydrogenase (Keitel et al. 2000) and quinohemoproteins type II ADH (Chen et al. 2002; Oubrie et al. 2002). The sixfold -propeller of s-GDH is observed in neuraminidase from influenza virus (Varghese et al. 1983) and bacterial sialidase (Crennell et al. 1993). Besides eightfold and sixfold -propellers, sevenfold -propeller structure is characteristic of quinoprotein methylamine dehydrogenase (which contains TTQ cofactor; Vellieux et al. 1989), the subunit of quinohemoprotein amine dehydrogenase (with CTQ cofactor; Datta et al. 2000), G-proteins (Wall et al. 1995; Lambright et al. 1996), phospholipase D (Stukey and Dixon 1999), copper-containing galactose oxidase (Ito et al. 1994), the human regulator of chromosome condensation (Renault et al. 1998), the integrin -subunit (Springer 1997), and the N-terminal domain of the clathrin heavy chain (ter Haar et al. 1998). A fourfold -propeller is described for hemopexin (Faber et al. 1995; Libson et al. 1995) and collagenase-3 structures (Li et al. 1995; Gomis et al. 1996).

On the basis of biochemical and kinetic data, hydride transfer and addition–elimination mechanisms have been proposed for MDH (Frank et al. 1988; Anthony 1996; Itoh et al. 1997) and sGDH (Olsthoorn and Duine 1998a; Dewanti and Duine 2000). The hydride transfer mechanism involves a general-base-catalyzed proton abstraction and hydride equivalent transfer from substrate to the quinone carbonyl carbon C5 of PQQ, and subsequent tautomerization of the C5 reduced PQQ to the hydroquinone (Fig. 1A,C). The addition–elimination mechanism involves nucleophilic addition of substrate to the C5 carbon of PQQ assisted by general-base catalysis of proton abstraction from substrate, followed by formation of covalent hemiketal intermediate and subsequent product elimination (Fig. 1B,D). The rate-limiting step for MDH and sGDH is conversion of the enzyme–substrate complex into the reduced coenzyme and product (Frank et al. 1988; Olsthoorn and Duine 1998a). The large deuterium isotope effect (~6) during the reductive phase of reaction indicates that both mechanisms are probable. The ease of nucleophilic addition to the C5 carbon of isolated PQQ by methanol, urea, ammonia, and amines has encouraged the assumption of formation of a covalent C5 PQQ–substrate complex in the initial step, characteristic of an addition–elimination reaction (Dekker et al. 1982; Itoh et al. 1993). A crystal structure report on methylhydrazine bound to sGDH indicated formation of a covalent C5 PQQ–inhibitor complex (Oubrie et al. 1999a). However, the electron density obtained for the X-ray structure of C5-reduced PQQ bound to MDH (Zheng et al. 2001) and the -anomer configuration of glucose in sGDH complex (Oubrie et al. 1999c) conforms to the hydride transfer mechanism (Fig. 1A,C). Theoretical studies have reported that the hydride transfer occurs from methanol to the O4 oxygen of PQQ (Zheng and Bruice 1997) and in another, from methanol to the O5 of PQQ (Jongejan et al. 2001). In view of the above reports and to understand better the role of enzyme active-site residues and catalytic mechanisms, we discuss 3-nsec SBMD studies by comparing MDH • PQQ • methanol, MDH • PQQH^–, and MDH • PQQH with sGDH • PQQ • glucose, sGDH • PQQH^– • glucolactone, and sGDH • PQQH structures.

View larger version (29K): [in this window] [in a new window]   Figure 1. Proposed enzyme mechanisms for methanol oxidation by MDH: (A) hydride transfer and (B) addition–elimination mechanisms. Proposed enzyme mechanisms for glucose oxidation by sGDH: (C) hydride transfer and (D) addition–elimination mechanisms.

	Molecular modeling procedures

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

The 1.9 Å sGDH • PQQH₂ • glucose complex with 600 crystal waters, PDB entry 1CQ1 [PDB] (Oubrie et al. 1999c) was chosen as the initial structure for MD studies on sGDH. The homodimer sGDH has 450 amino acid residues in each subunit. The cofactor in the X-ray structure was identified as hydroquinone, PQQH₂ with planar C5 and C4 atoms. The 1CQ1 [PDB] coordinates were therefore used to simulate the reactant complex sGDH • PQQ • glucose. For MD simulations on the sGDH • PQQH^– • glucolactone, the generated PQQH^– and glucolactone were exchanged for PQQ and glucose, respectively, in the X-ray structure. For MD studies on sGDH • PQQH, the generated PQQH was used.

The charges of PQQ, PQQH^–, PQQH, and glucolactone were evaluated using the ab initio procedures, which involve geometry optimization at the HF/6–31+G(d,p) level and evaluation of electrostatic potential at the MP2/6–31+G(d,p) level. In each structure the protonation sites of histidine residues were identified appropriately and the hydrogen atoms were generated. Based on the proposed hydride transfer mechanism (Fig. 1A,C), the carboxyl moiety of Asp 297 in MDH • PQQH^– and the imidazole of His 144 in sGDH • PQQH^– • glucolactone structures were protonated. The systems were solvated in an equilibrated TIP3P water sphere of 42 Å radii, using the center of mass of the cofactor as the origin. All the systems were minimized before, and after overlaying the explicit water molecules. Appropriate procedures of stochastic boundary molecular dynamics (Brooks and Karplus 1989) were followed. As per SBMD method, each system was partitioned into reaction (0–40.0 Å), buffer (41.01–42.0 Å), and reservoir (beyond 42.0 Å) regions. Newtonian dynamics treats the reaction region or the localized region of interest, whereas Langevin dynamics was applied to simulate the unwanted degrees of freedom of the buffer region by stochastic and mean forces. Details of the methodologies were published elsewhere (Reddy and Bruice 2003, 2004).

	Comparison of MDH and sGDH structures

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
Axial interactions of the active site
Axial interactions above and below the cofactor plane are mostly hydrophobic in nature (Fig. 2A,B). In MDH, the cofactor is buried in a chamber close to the center of the -propeller fold of each heavy subunit. The PQQ is sandwiched between the indole ring of Trp 237 and the vicinal disulfide Cys 103–Cys 104 (Fig. 2A). The vicinal disulfide of MDH has an unusual trans configuration for the peptide bond (212° in the MD and 190° in the X-ray structures). The strain caused by this unusual conformation of the peptide bond is alleviated by deviations of neighboring residue conformations from the generally preferred values, that is, (gauche⁺ instead of gauche^–) of Cys 103, (gauche⁺ instead of trans or gauche^–), and (trans instead of gauche^–or gauche⁺) of Asp 105. The active-site vicinal disulfide of MDH has also been reported in other PQQ enzymes, including ethanol dehydrogenase (Keitel et al. 2000), quinohemoproteins of type II ADH (Chen et al. 2002; Oubrie et al. 2002), and mercuric ion reductase (Schiering et al. 1991). The role of vicinal disulfide in enzyme catalysis is not clear and has been suggested to act as a shield from the bulk solvent at the entrance to the active site (Avezoux et al. 1995). MD investigations have established that the methyl group of methanol is encompassed by a hydrophobic cavity provided by the vicinal disulfide Cys 103–Cys 104, Trp 259, Trp 531, and Leu 547 (Fig. 2A). This region in the active site is adjacent to a more polar region containing such entities as Asp 297–CO₂^– and Glu 171–CO₂^–. Such positioning of methanol is consistent with X-ray structural predictions (Xia et al. 1999).

View larger version (64K): [in this window] [in a new window]   Figure 2. Active site of the MD averaged structures showing the axial interactions: (A) MDH • PQQ • methanol and (B) sGDH • PQQ • glucose, and the equatorial interactions: (C) MDH • PQQH and (D) sGDH • PQQH. In A and B, the carbons of PQQ, glucose, and the hydrophobic residues are shown in green, gray, and yellow, respectively. Notice in A that the substrate methanol is in a cavity surrounded by hydrophobic residues, above the PQQ. The nonbonded interactions in C and D are shown by red dashed lines. Notice that Arg 324 in C and Arg 228 in D are hydrogen-bonded to the O5 and O4 oxygens of PQQH. The amide side chains of Asn 387 in C and Asn 229 in D, respectively, are hydrogen-bonded to the O4 of PQQH.

Equatorial interactions of the active site
In the plane of the cofactor, various hydrogen-bond interactions occur between the cofactor and enzyme residues. Besides Ca²⁺ coordination, and other essential interactions that promote catalytic reaction in the active site (discussed in the following sections), there is a network of hydrogen-bonding interactions and salt bridges between the 2, 7, and 9 carboxylate oxygens of PQQH and enzyme residues (Fig. 2C,D). Such interactions of MDH • PQQH involve the C2 carboxyl group with side chains of Trp 467, Glu 55, and Arg 109 and the C9 carboxylate with side chains of Arg 109, Thr 153, and Ser 168 (Fig. 2C). The O7B oxygen at the C7 of PQQH interacts with Thr 235 and the backbone amide of Gly 169 and Ala 170 of MDH. In sGDH • PQQH structure, the C2, C7, and C9 carboxylates of PQQH make ion-pair interactions with Arg 408, Lys 377, and Arg 406, respectively (Fig. 2D). Most of the nonbonded separations related to these interactions of the MD structures are consistent with corresponding crystal structures of sGDH and MDH (Oubrie et al. 1999c; Zheng et al. 2001).

Active-site Ca²⁺ coordination
The enzymes MDH and sGDH are inactive in the absence of the Ca²⁺ ion (Adachi et al. 1990; Richardson and Anthony 1992; Olsthoorn et al. 1997). The Ca²⁺ ion is not involved in the binding of methanol or glucose. It is likely to play a role in the polarization of the C5 = O5 bond of PQQ along with the active-site Arg residue in MDH and sGDH (Anthony et al. 1994; Zheng and Bruice 1997; Olsthoorn and Duine 1998a). In the X-ray structures of MDH and sGDH, Ca²⁺ binds to the same cofactor ligands, the C7 carboxylate oxygen, the N6 pyridine nitrogen, and the C5 carbonyl oxygen (Oubrie et al. 1999c; Zheng et al. 2001). The other ligands of Ca²⁺ are different, provided by the side chains of Glu 171 and Asn 255 of MDH (Fig. 3B), whereas with sGDH, the ligands are main-chain carbonyl oxygen atoms of Gly 246 and Pro 247 and two water molecules (Wat55 and Wat89; Fig. 3D).

View larger version (46K): [in this window] [in a new window]   Figure 3. Ca2+ coordination in the MD averaged structures. (A) MDH • PQQ • methanol, (B) MDH • PQQH–, (C) MDH • PQQH, (D) sGDH • PQQ • glucose, (E) sGDH • PQQH– • glucolactone, and (F) sGDH • PQQH. The distances are given in angstroms. The values in brackets correspond to the crystal structure.

In the sGDH • PQQ • glucose MD complex, Ca²⁺ shows hepta-coordination similar to the X-ray structure (Fig. 3D; Oubrie et al. 1999c). In the sGDH • PQQH^– structure, the coordination to Pro 247 is absent, changing from hepta- to hexa-coordination of Ca²⁺ (Fig. 3E). In the sGDH • PQQH structure, Ca²⁺ coordination to the O5 protonated oxygen of PQQH is exchanged for a TIP3P (Jorgensen et al. 1983) water oxygen, which comprises the explicit solvent in which the enzyme is immersed during MD procedures (Fig. 3F). Absence of Ca²⁺ coordination to the O5 oxygen of PQQH is observed in the MD structures of MDH and sGDH. The oxyanion O5 of PQQH^– is a strong base and, as expected, remains coordinated to Ca²⁺ until protonated (Fig. 3B,E).

	Hydride transfer mechanism of MDH

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
Step 1: The oxidation of methanol by MDH is characterized by a general base abstraction of the ionizable proton of methanol, concerted with hydride equivalent transfer from the putative methoxide to the C5 quinone carbonyl carbon of PQQ (Fig. 1A). Because there is no experimental evidence for or against hydrogen tunneling, we simply use the form hydride transfer. It had been suggested that Asp 297–CO₂^– of MDH acts as the general base in this reaction (Anthony 1996; Xia et al. 1999; Keitel et al. 2000). Our MD studies on MDH • PQQ • methanol structure have shown that Asp 297–CO₂^– is too distant (5.54 ± 0.62 Å) from the hydroxyl oxygen of methanol to act as the general base (Reddy and Bruice 2003). The kinetic pH optimum for enzyme activity is 9.0, such that the MDH • PQQ • methanol and MDH • PQQH structures with both Glu 171 and Asp 297 as carboxylates represent the stable entities. It has been reported that the pH optimum is 7.0 for the enzyme reaction with cytochrome electron acceptor (Anthony 1992). We would expect the carboxylate entities in the active site would be ionized at either pH 7.0 or pH 9.0.

Glu 171–CO₂^– is the only functional base in the active site positioned to be the general base catalyst. One of the carboxylate oxygens of Glu 171–CO₂^– is at a distance 2.79 ± 0.16 Å from the methanol oxygen (Fig. 4A). The proton abstraction from the hydroxyl of methanol by Glu 171–CO₂^– is in concert with the hydride transfer, as the methanol carbon is at a distance of 3.77 ± 0.29 Å from the C5 carbon of PQQ. The hydrogen-bond interactions of the guanido nitrogens (NH1 and NH2) of Arg 324 with > C5 = O5 (2.81 ± 0.14 Å) and > C4 = O4 (2.93 ± 0.18 Å) oxygens are in position to aid PQQ reduction. Also, the amide side-chain nitrogen of Asn 387 is proximal (3.15 ± 0.25 Å) to the O4 oxygen of PQQ. The crystal water Wat1 forms a strong hydrogen bond with the O5 quinone carbonyl oxygen (2.71 ± 0.14 Å) and Asp 297–CO₂^– (2.75 ± 0.14 Å).

View larger version (33K): [in this window] [in a new window]   Figure 4. Concise view of the active site of the MD averaged structures. (A) MDH • PQQ • methanol, (B) MDH • PQQH–, (C) MDH • PQQH, (D) sGDH • PQQ • glucose, (E) sGDH • PQQH– • glucolactone, and (F) sGDH • PQQH. Nonbonded interactions are shown by red dashed lines and the Ca2+ ion is in green. The values correspond to distances (Å) between heavy atoms, and those in blue involve hydrogens.

Ammonia or methylamine has been used as an activator for MDH (from Hyphomicrobium X) with the phenazine ethosulphate dye-linked assay system (as artificial electron acceptor) at pH optimum 9.0 (Frank et al. 1988). Stopped-flow kinetics studies on MDH indicated that a high concentration of ammonia acts as an inhibitor (Harris and Davidson 1993b). Also, it has been demonstrated that ammonia has little kinetic effect in the presence of physiological cytochrome electron acceptor (Dijkstra et al. 1989). In view of these, MD simulations undertaken on the MDH • PQQ • methanol structure with NH₃ have shown that NH₃ diffuses away from the active site, interacting with the solvent at the surface. In any case, NH₃ at pH 7.0 would exist as NH₄⁺. In contrast, NH₄⁺ remains in the active site during the simulation of the MDH • PQQ • methanol • NH₄⁺ structure, making hydrogen-bond interactions with the O5 oxygen of PQQ, Asp 297–CO₂^–, and Glu 171–CO₂^–. However, methanol moves to the entrance of the enzyme active site, near the indole nitrogen of Trp 531 and the Cys 103–Cys 104 vicinal disulfide. Possibly, NH₃ interacts with MDH at a different position, which does not disturb the environment of the active site (Harris and Davidson 1993b). Work is in progress to study the effect of imino and amino moieties substituted, instead of the quinone carbonyl oxygen O5 of PQQ in the MDH • PQQ • methanol structure.

The immediate product from methanol oxidation by MDH is formaldehyde. The means of departure of O = CH₂ is unknown. It is possible that O = CH₂ interacts with one of the many waters in the active-site vicinity, to form the stable hydrate (HO)₂CH₂.

Step 2: Hydride transfer to –C5(= O) – of PQQ leads to – (H)C5(O^–) – of PQQH^–. Thus, Glu 171–CO₂^–, a general base, was examined to see whether once protonated it acts as a general acid species to protonate the oxyanion O5 of PQQH^–. MD simulations establish that Glu 171–CO₂H formed during hydride transfer is not placed in a position to protonate the oxyanion – (H)C5(O^–) – moiety of PQQH^– (Reddy and Bruice 2003). It could be imagined that Glu 171–CO₂H transfers its proton to Asp 297–CO₂^–, which would allow the MDH • PQQH^– complex to be a transient structure. During MD simulations, the average distance from the oxyanion O5 of PQQH^– and the protonated carboxylate oxygen of Asp 297–CO₂H is 2.57 ± 0.08 Å (Fig. 4B), indicating that Asp 297–CO₂H could act as the required proton donor (Reddy et al. 2003). This feature is not obvious in the crystal structure because of a large separation (4.07 Å) between the carboxyl oxygen of Asp 297 and the O5 oxygen of the C5 reduced PQQ (Zheng et al. 2001). A hydrogen bond between the oxyanion O5 of PQQH^– and Wat1 (2.91 ± 0.27 Å) is observed. These interactions render the O5^– oxygen to move farther from the guanido nitrogen NH1 of Arg 324, in contrast to the observed hydrogen bond (3.03 Å) in the X-ray structure. The guanido nitrogen NH1 of Arg 324 (3.27 ± 0.22 Å) is hydrogen-bonded to Asp 297–CO₂H. The O4 carbonyl oxygen at C4 of PQQH^– is involved in bidentate interactions with the guanido side chains of Arg 324 (2.73 ± 0.09 Å and 2.90 ± 0.16 Å), consistent with the crystal structure (Zheng et al. 2001). These interactions are expected to assist rearrangement of PQQH^– PQQH.

Step 3: Asp 297–CO₂^– has been suggested (Anthony 1996; Xia et al. 1999) to assist as a general base in the transfer of the H5 hydrogen at tetrahedral C5 of PQQH to provide the hydroquinone PQQH₂ (Fig. 1A). In the MD structure of MDH • PQQH, Asp 297–CO₂^– is at a distance of 3.43 ± 0.44 Å, and is not poised to assist in migration of the H5 hydrogen of PQQH. This is because Asp 297–CO₂^– coordinated to Ca²⁺ is also preoccupied in hydrogen-bond interactions with the O5 protonated oxygen of PQQH (2.98 ± 0.26 Å) and Wat1 (2.64 ± 0.09 Å and 2.87 ± 0.12 Å; Fig. 4C).

The average distance between the carboxylate oxygen of Glu 171–CO₂^– and H5 at the tetrahedral C5 of PQQH is large (3.68 ± 0.26 Å). So Glu 171–CO₂^– is unlikely to aid in proton isomerization of – (H)C5(OH) –C4( = O) – –C5(OH) = C4(OH) –. This reaction is very exergonic because of the gain in aromaticity. Thus, any catalysis of the rearrangement would amount to a solvation of the transferring proton. Interestingly, MD simulations suggest that a Glu 171–CO₂^– ... H₂O ... H–C5 relay is involved in the solvation of the proton transfer. The water molecule involved can be either Wat97 or TIP3P, which exchanges during MD simulation. This is an interesting feature not noticed in the X-ray structures (Ghosh et al. 1995; Xia et al. 1999; Zheng et al. 2001). The reaction is assisted by hydrogen-bonding of the guanido nitrogens of Arg 324 with the O5 (2.86 ± 0.16 Å) and O4 oxygens (3.15 ± 0.33 Å) of PQQH (Fig. 4C). In the MDH • PQQH structure, Wat1 is hydrogen-bonded (2.88 ± 0.17 Å) to the O5 protonated oxygen of PQQH in good agreement with the crystal structure (Zheng et al. 2001). The hydrogen-bonding between NH1–HH12 (Arg 324) and the carboxylate oxygen (OD1) of Asp 297–CO₂^– is absent.

	Addition–elimination mechanism of MDH

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
The proposed hydride transfer and addition–elimination mechanisms for methanol oxidation by MDH (Frank et al. 1988; Anthony 1996; Itoh et al. 1997) and glucose oxidation by sGDH (Olsthoorn and Duine 1998a; Dewanti and Duine 2000) involve the concerted general-base catalysis of proton abstraction from substrate (Fig. 1). The addition–elimination mechanism is characterized by nucleophilic addition of methanol to the C5 of PQQ (Fig. 1B). For nucleophilic addition to occur, the methanol oxygen has to be proximal to the C5 of PQQ. A time variation plot of nonbonded distance between the C5 of PQQ and methanol oxygen is given in Figure 5A, which shows the C5(PQQ) ... O(methanol) value is very large (4.59 ± 0.26 Å) for the additional elimination reaction to occur (see also Fig. 4A). This feature is in good agreement with the interpretation of the X-ray structure that the C5-reduced PQQ bound to MDH can occur only as a PQQ–hydride adduct (Zheng et al. 2001).

View larger version (51K): [in this window] [in a new window]   Figure 5. Time variation plots related to the addition–elimination mechanism of MDH and sGDH. (A) Distance between the C5 of PQQ and methanol oxygen and (B) distance between the C5 of PQQ and the O1 oxygen of glucose.

	Hydride transfer mechanism of sGDH

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

In the X-ray structure, the C1 glucose atom is directly above the C5 of PQQ, at a distance within 3.2 Å (Oubrie et al. 1999c). In -D-glucose, the C1 hydrogen atom is axially positioned and points down, in the direction of the C5 of PQQ, at a separation of 1.2 Å from the latter. Therefore, the demonstrated absolute -anomer specificity for glucose supports hydride transfer of the H1 at C1 of glucose to the C5 carbon of PQQ (Olsthoorn and Duine 1998b; Oubrie et al. 1999c). Such geometry of substrate and cofactor active-site groups has been observed in nicotinamide (Westheimer 1987; Karplus and Schulz 1989; Ramaswamy et al. 1994) and flavin-dependent oxidoreductases (Mattevi et al. 1996), which also use hydride equivalent transfer mechanisms.

Step 2: It has been suggested (Olsthoorn and Duine 1998b; Dewanti and Duine 2000) that the imidazolium of His 144 donates a proton to the oxyanion O5 of PQQH^– (Fig. 1B). In the sGDH • PQQH^– • glucolactone structure, the average distance from the O5 of PQQH^– and His 144–ImH⁺ is 4.79 Å. Thus, protonation of the oxyanion entity of PQQH^– by His 144 is not possible. The immediate product from glucose oxidation is glucolactone, which interacts with Arg 228 and His 144 of sGDH. The O5 of PQQH^– is engaged in hydrogen-bond interactions with the guanido nitrogens of Arg 228 (2.62 ± 0.08 Å and 2.86 ± 0.23 Å) and Wat89 (3.14 ± 0.18 Å; Fig. 4E). The protonated NE2 of His 144 forms a hydrogen bond (3.05 ± 0.23 Å) with the O4 carbonyl oxygen of PQQH^–. It is therefore plausible that the rearrangement of – (H)C5(O^–) – C4( = O) – of PQQH^– to –C5(OH) = C4(OH) – of PQQH₂ is assisted by general acid protonation of the > C4 = O oxygen by His 144–ImH⁺ and hydrogen bonds of Arg 228 to the oxyanion O5. Also, the imidazolium of His 144 is proximal (3.27 ± 0.55 Å) to the O1 oxygen of the glucolactone product.

Step 3: His 144 has been suggested (Olsthoorn and Duine 1998b; Dewanti and Duine 2000) to play the role of general base in migration of the H5 hydrogen at the tetrahedral C5 of PQQH to provide the hydroquinone PQQH₂ (Fig. 1B). This role for His 144 in the sGDH • PQQH structure is unlikely because the NE2 of His 144 is too far (4.82 ± 0.25 Å) from the H5 of PQQH (Fig. 4F). According to the MD studies, any rearrangement of – (H)C5(OH) – C4( = O) – of PQQH to –C5(OH) = C4(OH) – of PQQH₂ would be assisted by two hydrogen-bond networks mediated by waters Wat55 and Wat89. These involve Asp 252 and Gln 246 through Wat55, and Arg 228 and His 144 through Wat89 (Reddy and Bruice 2004). Wat89, at a distance of 2.74 ± 0.19 Å from the H5 of PQQH, would be involved in the departure of the C5 hydrogen of PQQH, if this species should be intermediate to PQQH₂. This is an interesting feature not suggested by X-ray studies (Oubrie et al. 1999c). The hydrogen-bond interactions of the guanido nitrogens of Arg 228 with the O5 (2.97 ± 0.18 Å) and O4 (2.94 ± 0.15 Å) oxygens of PQQH would have a positive influence on the hydroquinone formation (Fig. 4F).

	Addition–elimination mechanism of sGDH

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
The oxidation of glucose by sGDH via the proposed addition–elimination mechanism (Olsthoorn and Duine 1998a; Dewanti and Duine 2000) is allowed when the O1 oxygen of glucose makes a nucleophilic addition to the C5 carbon of PQQ concerted with general-base catalysis of proton abstraction from the O1 hydroxyl of glucose (Fig. 1D). A time variation plot of nonbonded distance between the C5 carbon of PQQ and the O1 oxygen of glucose is given in Figure 5B. The O1 of glucose points in a direction away from the C5 of PQQ (Fig. 4D). This is characteristic of -anomer configuration, with the hydrogen at C1 pointing below, directly toward the C5 of PQQ (which facilitates hydride transfer; Oubrie et al. 1999c). The average distance between the C5 of PQQ and the O1 of glucose is large (3.46 ± 0.26 Å) for adequate juxtaposition (based on van der Waals separation), compared with the distance between the C5 of PQQ and the C1 of glucose (3.57 ± 0.17 Å). The results are consistent with the proposal that considerable rearrangements in the active site are required to form a covalent C5 PQQ–glucose complex (Oubrie et al. 1999c; Oubrie and Dijkstra 2000). Therefore, the addition–elimination mechanism is not preferred for glucose oxidation by sGDH.

	Conclusions

TOP Abstract Introduction Molecular modeling procedures Comparison of MDH and... Hydride transfer mechanism of... Addition-elimination mechanism... Hydride transfer mechanism of... Addition-elimination mechanism... Conclusions References

 
Our investigations indicate that the orientation of the enzyme active-site residues of MDH and sGDH catalyze their respective redox reactions via the same hydride transfer mechanism (Figs. 6, 7) and not by a covalent addition–elimination mechanism. The reaction is initiated by abstraction of a proton from the substrate by the general base Glu 171–CO₂^– in the MDH • PQQ • methanol complex, and His 144 assisted by Asp 163–CO₂^– in the sGDH • PQQ • glucose structure, in concert with the direct hydride transfer from substrate to the C5 quinone carbonyl carbon of PQQ. In the MDH • PQQH^– structure, Asp 297–CO₂H acts as a proton source to the oxyanion O5 of PQQH^– to form PQQH (Fig. 6). In the sGDH • PQQH^– • glucolactone structure, the imidazolium of His 144 is unlikely to be a proton source of the oxyanion O5 of PQQH^– (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Figure 6. Plausible mechanism of methanol oxidation by MDH from MD studies. For actual positioning, see Figure 4, A–C.

View larger version (23K): [in this window] [in a new window]   Figure 7. Functional groups positioned to explain the mechanism of glucose oxidation by sGDH (from MD studies). For actual positioning, see Figure 4, D–E.

The presence of Arg 324 and Asn 387 residues in MDH and Arg 228 and Asn 229 in sGDH close to the cofactor in the active sites is conserved. But the precise orientation of these residues with respect to the cofactor is slightly different in both enzymes. The position of the Arg residue is stabilized by proximal water (either Wat124 in MDH or Wat89 in sGDH). In both enzymes, hydrogen-bondings to Arg 324 of MDH and Arg 228 in sGDH polarizes the > C5 = O5 of PQQ and the tetrahedral C5–O5 of PQQH^–, and PQQH to promote the reaction. The hydrogen-bonding of the amide nitrogen of Asn 387 of MDH or Asn 229 of sGDH to the O4 carbonyl oxygen of the cofactor may also aid the reaction. However, the interactions of Arg 324 and Asn 387 with the O5 and O4 of PQQH^–, respectively, are absent in the MDH • PQQH^– structure. Bidentate hydrogen-bond interactions of Arg 324 with the O4 oxygen of PQQH^–stabilize MDH • PQQH^–, but similar interactions of Arg 228 with the O5 oxyanion of PQQH^– are dominant in the sGDH • PQQH^– • glucolactone structure.

The hydrogen-bonding of the O5 oxygen of cofactor to Wat1 of MDH or Wat89 of sGDH is observed during the entire reaction in both enzymes. Distinctly, Ca²⁺ coordination to the O5 quinone carbonyl oxygen of PQQ is absent in the MDH • PQQ • methanol structure, whereas the same is observed in the sGDH • PQQ • glucose structure. In both enzymes, the ligands of Ca²⁺ to PQQH^– (in MDH • PQQH^–and sGDH • PQQH^– • glucolactone) and PQQH (in MDH • PQQH and sGDH • PQQH) are the same. In sGDH, the hydrogen-bondings of Wat89 to the guanido of Arg 228 are observed throughout the entire reaction. In the MD structures of sGDH, the orientation of His 144 and Asn 229 residues are stabilized by hydrogen-bonding interactions with Asp 163–CO₂^– and the hydroxyl oxygen of Ser 146, respectively, in agreement with the X-ray structure (Oubrie et al. 1999c).

Various new structural and mechanistic features of MDH and sGDH not amenable for experimentation have been explored using MD studies. Our studies provide evidence that MD procedures have the ability to clarify enzyme mechanisms, based on juxtaposition of the active-site residues. Is the hydride transfer mechanism a common feature of PQQ enzymes and other quinoproteins? It is early to answer conclusively, as more computational work is required on other PQQ dehydrogenases, and quinoproteins involving amino-acid-derived cofactors. Future MD studies on quinoproteins should be promising in providing valuable insights on structure and enzymatic mechanisms.

	Acknowledgments

 
This work was supported by NIH (5R37DK0917138) and NSF (MCB-9727937) grants.

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