Molecular Determinants of the Regioselectivity of Toluene/o … · Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli Federico II, Complesso Universitario di

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 823–836 Vol. 75, No. 30099-2240/09/$08.00�0 doi:10.1128/AEM.01951-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Determinants of the Regioselectivity of Toluene/o-XyleneMonooxygenase from Pseudomonas sp. Strain OX1�†

Eugenio Notomista,1,2‡* Valeria Cafaro,1‡ Giuseppe Bozza,1 and Alberto Di Donato1,3

Dipartimento di Biologia Strutturale e Funzionale, Universita di Napoli Federico II, Complesso Universitario di Monte S. Angelo,Via Cinthia 4, 80126 Naples, Italy,1 and Facolta di Scienze Biotecnologiche, Universita di Napoli Federico II,2 and

CEINGE-Biotecnologie Avanzate S.c.ar.l.,3 Naples, Italy

Received 21 August 2008/Accepted 28 November 2008

Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenaseswhich share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMMpossesses defined substrate specificity and regioselectivity which match the metabolic requirements of thestrain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, andp-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture ofo-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, weprepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Usingthe Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate com-plexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutantToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes anda simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regio-selectivities of the majority of the variant proteins with good accuracy. The results show not only that thefine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active sitebut also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.

Bacterial multicomponent monooxygenases (BMMs) are alarge and heterogeneous family of nonheme di-iron enzymeswhich share the very interesting ability to activate dioxygen andtransfer a single oxygen atom to a wide variety of substrates(13, 23). Aliphatic and aromatic hydrocarbons are converted,respectively, into alcohols and phenols (3, 6, 15, 21, 22, 38),alkenes are converted into epoxides (8), and sulfur-containingcompounds are oxidized into sulfoxides and sulfones (10).

As BMMs allow bacteria to grow on hydrocarbons or xeno-biotics as the sole source of carbon and energy, several mem-bers of this protein family, including the soluble methanemonooxygenases (MMOs) (15, 21), alkene monooxygenases(8), phenol hydroxylases (PHs)/toluene 2-monooxygenases(T2MOs) (3, 22), and toluene monooxygenases (TMOs) suchas toluene 4-monooxygenase (T4MO) from Pseudomonas men-docina KR1 (38) and toluene/o-xylene monooxygenase(ToMO) from Pseudomonas sp. strain OX1 (6), have beencharacterized thoroughly.

All these enzymes possess defined substrate specificity, re-gioselectivity, and enantioselectivity properties. For example,TMOs and PHs perform two consecutive hydroxylation reac-tions with aromatic rings, but usually TMOs are more efficientin the first hydroxylation step, whereas PHs are more efficient

in the second (3, 5). Moreover, each TMO and PH shows itsown characteristic regioselectivity. T4MO produces more than96% p-cresol from toluene (25), whereas ToMO produces amixture of the three isomers of cresol (5). PHs usually producea large excess of o-cresol—70 and 90% in the cases of PH fromPseudomonas sp. strain OX1 (5) and T2MO from Burkholderiacepacia G4 (22), respectively.

Thus, it appears that the BMM family constitutes an archiveof powerful catalysts that could be used to construct new cat-alysts for the bioremediation of environmentally harmful sub-stances and for industrial biosyntheses. Certainly, an under-standing of the molecular determinants of BMM substratespecificity, regioselectivity and enantioselectivity properties ispreliminary to the rational design of new, improved catalysts,as proved by the large number of studies on BMM catalyticmechanisms and on synthetic analogues capable of catalyzingreactions similar to those catalyzed by BMMs (14, 28, 39).

The results of several structural and functional studies sug-gest that the catalytic mechanisms of BMMs are very similar(13, 19). The major subunit (A in TMOs and � in MMOs) ofthe hydroxylase component (the H complex) contains a di-ironcluster bound to four glutamate and two histidine residues.These residues, and several other conserved hydrophilic resi-dues, form an H bond network on one side of the iron ions (13,19). On the other side, nonconserved hydrophobic residuesform the substrate binding pocket (13, 19). The catalyticallyactive diferrous form, interacting with dioxygen, produces adi-iron(III) intermediate (the peroxo intermediate) which, atleast in the case of MMOs, turns into a di-iron(IV) formknown as diamond core (19). The peroxo and diamond coreintermediates each transfer one oxygen atom to the substrate(19). The possible intermediates involved in the transfer of

* Corresponding author: Dipartimento di Biologia Strutturale eFunzionale, Universita di Napoli Federico II, Complesso Universitariodi Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy. Phone: 39-081-679208. Fax: 39-081-679313. E-mail: [email protected].

‡ Valeria Cafaro and Eugenio Notomista contributed equally to thepaper.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 12 December 2008.

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oxygen to the aromatic ring (16, 19, 20) are shown in Fig. 1.The reactive species, likely a di-iron(III) intermediate, attacksthe pi-electron system of the aromatic ring, forming epoxide 1or delocalized carbocation 2. The opening of the epoxide ringeventually provides the delocalized carbocation. The migrationof a hydride from the sp3-hybridized carbon to the adjacentatom then converts the carbocation to the more stable ketone3. Finally, the dissociation of the ketone and its tautomeriza-tion yield phenolic product 4.

The different regioselectivities shown by BMMs have beenattributed previously to differences in the shape of the activesite pocket, an idea supported by the fact that several pointmutations in the active sites of T4MO, toluene p-monooxygen-ase, ToMO, MMO, and B. cepacia G4 T2MO cause largevariations in the regioselectivities of these BMMs (2, 7, 26, 30,34, 35). In a previous paper (4), we have reported that muta-tions at position 103 of the A subunit change the regioselec-tivity of ToMO from Pseudomonas sp. strain OX1. However, acomplete study of the active site pocket of ToMO A is stilllacking.

In this paper, we report the effects of the substitution of sixresidues in the ToMO A active site on substrate specificity andregioselectivity. Furthermore, we present a detailed analysis ofthe molecular determinants of regioselectivity based on thedocking of substrates and hypothetical intermediates of thearomatic hydroxylation reaction into the active site of the crys-tallographic structure of ToMO (Protein Data Bank [PDB]code 1T0Q [32]), followed by a Monte Carlo optimization. Theresults show that (i) the fine-tuning of TMO regioselectivitycan be achieved through a careful alteration of the shape of theactive site pocket and that (ii) the effects of mutations onregioselectivity can be quantitatively predicted using the pro-cedure described herein.

MATERIALS AND METHODS

Materials. Bacterial cultures, plasmid purifications, and transformations wereperformed according to the procedures of Sambrook et al. (31). Escherichia colistrains JM109 and CJ236 and vector pET22b(�) were from Novagen. PlasmidpBZ1260 (1) used for the expression of the ToMO cluster was kindly supplied byP. Barbieri (Dipartimento di Biologia Strutturale e Funzionale, Universitadell’Insubria, Varese, Italy). E. coli strain JM101 was purchased from Boehr-inger. The pGEM-3Z expression vector, Wizard SV gel, and the PCR clean-upsystem for the elution of DNA fragments from agarose gel were obtained fromPromega. Enzymes and other reagents for DNA manipulation were from NewEngland Biolabs. The oligonucleotides were synthesized at MWG-Biotech

(Ebersberg, Germany). All other chemicals were from Sigma. The expressionand purification of recombinant catechol 2,3-dioxygenase from Pseudomonas sp.strain OX1 are described elsewhere (36).

ToMO A mutagenesis. Plasmids for the expression of ToMO complexes withmutated ToMO A subunits were prepared by site-directed mutagenesis of plas-mid pTOU as described previously (4). Sequences of the mutagenic oligonucle-otides are reported in Table S1 in the supplemental material.

Determination of apparent kinetic parameters and identification of products.Assays were performed as described previously (4, 5) using E. coli JM109 cellstransformed with plasmid pBZ1260 or plasmid pTOU, which expresses wild-typeToMO or ToMO mutant enzymes, respectively.

All kinetic parameters were determined using whole cells (4, 5). Enzymaticactivity on phenol was measured by monitoring the production of catechol incontinuous coupled assays with recombinant catechol 2,3-dioxygenase fromPseudomonas sp. strain OX1 (5). The determination of apparent kinetic param-eters for benzene, toluene, o-xylene, and naphthalene was carried out by adiscontinuous assay (5). For the calculation of the kcat values, amounts of pro-teins were calculated as described previously (5). All the ToMO mutant enzymesshowed expression levels similar to that of the wild-type enzyme.

Reaction products were identified as described previously (5). All the regio-specificity studies were performed using substrate concentrations higher than theKm values. Under these conditions, absolute yields of products were proportionalto kcat values.

Modeling of substrates and intermediates into the active site of ToMO A.Substrates and reaction intermediates were docked into the active site of ToMOA by using the Monte Carlo energy minimization strategy. The ZMM-MVMmolecular modeling package (ZMM Software Inc. [http://www.zmmsoft.com])was used for all calculations. This software allows conformational searches usinggeneralized coordinates such as torsion and bond angles instead of conventionalCartesian coordinates (40).

Atom-atom interactions were evaluated using assisted model building withenergy refinement force fields (37) with a cutoff distance of 8 Å. Conformationalenergy calculations included van der Waals, electrostatic, H bond, and torsioncomponents. A hydration component was not included. Electrostatic interactionswere assessed with a relative dielectric constant of 4.

Substrate and intermediate structures were prepared using the PyMOL soft-ware (DeLano Scientific LLC). Geometry was optimized using the Zl module ofZMM. Partial charges were attributed using the complete neglect of differentialoverlap method in the HyperChem software (HyperCube Inc. [http://www.hyper.com]).

The X-ray structure of the ToMO A-thioglycolate complex (PDB code 1T0Q)was used to build the models of the ToMO A-substrate and ToMO A-interme-diate complexes. To reduce computational time, a double-shell model of theenzyme was built. The inner shell included 28 ToMO A residues surrounding theactive site cavity. During energy calculation procedures, the side chain torsionangles—but not the backbone torsion angles—of these residues were allowed tovary. Due to the asymmetric shape of the cavity, which is flat with the di-ironcluster on one end and evolutionarily nonconserved hydrophobic residues on theopposite side, the residues of the inner shell were selected manually. Theyincluded the ligands of the iron ions, all residues with at least one side chain atomcontributing to the hydrophobic part of the active site cavity, and all residues withat least one side chain atom less than 5 Å from the previous residues.

The outer shell included 139 residues, which did not belong to the inner shell

FIG. 1. Possible intermediates in the aromatic hydroxylation reaction catalyzed by ToMO. Intermediates 1, 2, and 3 are an epoxide, acarbocation, and a ketone, respectively. R1 and R2 are hydrogen atoms or methyl groups. The geometrical features of the di-iron(III)-(hydro)per-oxide intermediate and the details of the O-O bond cleavage reaction are not known.

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and were located less than about 16 Å from the active site pocket. During energycalculation procedures, both the backbone and the side chain torsion angles ofthe outer shell residues were not allowed to vary.

To further restrict the conformational freedom of the iron cluster and of theprotein-ligand complexes, two flat-bottom parabolic penalty functions—the so-called constraints—available in ZMM were used. These functions increase theconformational energy of the system if it deviates from specified parameters. Theatom-atom distance constraint applies a force to the system when the distancebetween two specified atoms deviates from a specified value or interval. This typeof constraint was used to fix the distances between the two iron ions and betweeneach iron ion and the surrounding atoms, including the bridging water moleculeand the terminal water molecule. Atom-atom distance constraints were also usedto fix the conformation of the ligands of the di-iron cluster. A force constant of1,000 kcal/mol/Å was used. The atom-atomic coordinate constraint applies aforce to the system when an atom moves farther than a specified distance fromparticular Cartesian coordinates. This constrain was used to prevent the move-ment of the Oε1 atom of the Glu103 side chain more than 0.5 Å from the originalposition observed in the crystallographic structure.

A �2 charge was arbitrarily assigned to each iron atom, both to account forelectron density transfer from the ligands to iron ions and to avoid strongelectrostatic attractive and repulsive interactions with negative and positiveatoms, respectively, of substrates and intermediates. Similarly, both the bridg-ing and terminal solvent molecules observed in the 1T0Q structure were mod-eled as neutral water molecules rather than OH� ions. van der Waals radii ofiron ions and oxygen atoms of water molecules were arbitrarily set to 0.8 and 1.5Å, respectively, in order to reduce steric hindrance inside the di-iron cluster.

Complexes with total energies of up to 8 kcal/mol higher than that of thelowest-energy complex were stored for the analysis of energy contributions. Totalenergy was partitioned into intrareceptor, intraligand, and receptor-ligand ener-gies and energies of the constraints. Receptor-ligand energy was further parti-

tioned into van der Waals, electrostatic, and H bond components. Moreover,receptor-ligand energy was also partitioned (i) per active site residue in order toevaluate the contribution of each residue to the binding of ligands and (ii) perligand atom in order to evaluate the contributions of the ring and methyl sub-stituents. Intrareceptor energies gave an estimation of the energy costs forreceptor (ToMO A) conformational changes upon ligand binding.

The docking procedure is described in detail in the supplemental material. ThePDB files for the initial manually generated complexes and the ZMM instructionfiles containing the lists of mobile residues, constraints, and parameters usedduring calculations are available upon request.

RESULTS AND DISCUSSION

Kinetic model for ToMO regioselectivity. The results of sev-eral studies of BMMs suggest that the regioselectivities ofthese enzymes depend on the shape of the active site cavity,which is believed to influence the orientations of substratesand reaction intermediates during catalysis (4, 7, 9, 19, 26, 34).The ToMO active site pocket is buried deeply inside theToMO A subunit and is in contact with the surface through along tunnel whose diameter is large enough to allow the en-trance of substrates and the exit of products (32). The activesite cavity (Fig. 2A and B) shows a lens-like shape. The dis-tance between two atoms on opposite sides of the edge is about10.5 to 12.5 Å. Assuming a van der Waals radius of about 2 Å,these distances correspond to an inner diameter of 6.5 to 8.5 Å.

FIG. 2. Active site pocket of ToMO A. Panels A and B show the active site of the crystal structure of ToMO A (PDB code 1T0Q). Only residuescontributing to define the edge of the cavity are shown. In panel B, the grid cuts the cavity in such a way as to provide the largest section. Carbonatoms are shown in red (Glu134 and Glu231), green (Ala107), yellow (Met180), blue (Glu103), magenta (Phe176), cyan (Ile100), and orange(Phe205). (C) Superimposition of the PDB code 1T0Q crystal structure (colored as in panels A and B) onto the structure of the complexToMO-CCI 2 for the reaction leading to phenol production from benzene (carbon atoms are shown in white). (D) Superimposition of the complexToMO-CCI 2 (carbon atoms are shown in white) onto the complex ToMO-ketone 3 (carbon atoms are shown in green) for the reaction leadingto phenol production from benzene. THG, thioglycolate. CCI indicates CCI 2 and KTI indicates ketone 3.

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The distance between two atoms placed above and below theplane of the cavity is about 8.5 Å; hence, the thickness of thecavity is about 4.5 Å. Therefore, the cavity is slightly largerthan a benzene molecule, which has a thickness of about 4 Åand a diameter of about 6.4 Å. The grid in Fig. 2B cuts thecavity into two halves in such a way as to highlight the sectionwith the larger surface.

The edge of the pocket (Fig. 2A and B) is formed by residuesAla107, Met180, Glu103, Phe176, Ile100, and Phe205 and twoiron ligands, Glu134, adjacent to Ala107, and Glu231, adjacentto Phe205. It should be noted that in the case of Glu103, onlythe methylene groups contribute to the pocket (4, 32). Thr201and Phe196 form one of the faces of the cavity, whereasGlu104 forms the opposite one.

In a previous paper (4), we explained the regiospecificity ofToMO A for toluene by hypothesizing that there are at leastthree different positions in the active site pocket which canaccommodate the methyl group of toluene. These three sub-sites can orient the methyl group of the substrate such that itsortho, meta, or para carbon is presented to the di-iron center.Thus, it is the difference in the affinities of the subsites for thebinding of the methyl group which determines the relativeabundances of three different enzyme-substrate complexes,which can account for the observed distribution of cresolsproduced by ToMO. Using a manual docking procedure (4),we mapped an ortho subsite located among Ala107, Met180,and Glu103, a meta subsite among Glu103, Phe176, and Ile100,and a para subsite located between Ile100 and Phe205.

Figure 3 shows a new, more complex kinetic model ofToMO regioselectivity. According to model i, toluene wouldbind to the active site in three different catalytically productiveorientations, thus giving rise to three different enzyme-toluene(ET) complexes that lead to the production of o-, m-, andp-cresol isomers (complexes ETo, ETm, and ETp, respectively)through at least one enzyme-intermediate (EI) complex (EIo,EIm, or EIp, corresponding to o-, m-, or p-cresol, respectively).According to this model, the ET-EI conversion is the rate-limiting step. As the interactions between toluene and theactive site cavity are limited to van der Waals interactions andas the active site cavity is larger than the toluene molecule, itis likely that the interconversion of the ET complexes is fastwith respect to their transformation to cresols. The new modelcan be described by six equilibrium constants: Ko-m, Ko-p, andKm-p for the conversions ETo^ ETm, ETo^ ETp, and ETm^ETp, respectively, and K‡

o, K‡m, and K‡

p for the conversion ofeach productive ET complex to the corresponding transitionstate (ET‡) complex. Each ET‡ complex can turn into the othertwo activated complexes through the ET complexes. There-fore, we can define three equilibrium constants for the conver-sions ET‡

o ^ ET‡m, ET‡

o ^ ET‡p, and ET‡

m ^ ET‡p, which

will be the products of the constants defined above:

K‡o-m � �ET‡

m�/�ET‡o� � Ko-mK‡

m/K‡o � exp� � �G‡

o-m/RT�

(1)

K‡m-p � �ET‡

p�/�ET‡m� � Km-pK‡

p/K‡m � exp� � �G‡

m-p/RT�

(2)

K‡o-p � �ET‡

p�/�ET‡o� � Ko-mKm-pK‡

p/K‡o � exp� � �G‡

o-p/RT�

(3)

where �G‡o-m, �G‡

m-p, and �G‡o-p are the free-energy differ-

ences for the three conversions, R is the gas constant, and T isthe absolute temperature.

This new model also includes an undefined number of un-productive ET (ETu) complexes. It should be noted that thenumber and the stability of these unproductive complexes donot influence regioselectivity but only the apparent kcat value,as they decrease the relative abundances of the ETo, ETp, andETm complexes and of the corresponding ET‡ complexes atequilibrium.

Each ET‡ provides the corresponding EI, which in turnreleases a cresol isomer. Each ET‡-EI transformation shouldproceed with the same rate, , which is given by the followingwell-known relation: kT/h, where k and h are the Boltz-mann and Planck constants, respectively, and T is the absolutetemperature. According to model i (Fig. 3), the relative abun-dances of o-, m-, and p-cresol isomers produced by the enzymeare determined by the relative abundances of the three tran-sition state ET‡ complexes at equilibrium. Therefore, calculat-ing the energy differences, �G‡

o-m, �G‡m-p, and �G‡

o-p, shouldallow the prediction of the percentages of cresols formed.

Two components should contribute to these energy differ-ences: (i) the covalent bond energy (bE‡), which includes theenergy of the bonds among ligand atoms and between ligandand protein atoms, for example, those between the oxygenatom transferred to the substrate and each iron of the cluster

FIG. 3. Kinetic models for the hydroxylation reaction of tolueneand o-xylene. CCIs 5 to 12 are the CCIs deriving from toluene ando-xylene shown in Fig. 4. oC, mC, and pC, o-, m-, and p-cresols.



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(Fig. 1), and (ii) the ligand-protein noncovalent bond energy(nbE‡). Assuming that bE‡ is scarcely influenced by the positionof the substituent, the �G‡ values should depend essentially onthe nbE‡ contributions:

�G‡o-m � �nbE‡

m � nbE‡o� � �bE‡

m � bE‡o� � �nbE‡

m � nbE‡o�

� �nbE‡o-m (4)

�G‡m-p � �nbE‡

p � nbE‡m� � �bE‡

p � bE‡m� � �nbE‡

p � nbE‡m�

� �nbE‡m-p (5)

�G‡o-p � �nbE‡

p � nbE‡o� � �bE‡

p � bE‡o� � �nbE‡

p � nbE‡o�

� �nbE‡o-p (6)

The kinetic model ii shown in Fig. 3 illustrates the case ofo-xylene. Two productive enzyme-substrate complexes (re-ferred to hereinafter as EX complexes, where X represents thesubstrate)—EX2,3 and EX3,4—and the corresponding EX‡

complexes yield 2,3-dimethylphenol (2,3-DMP) and 3,4-DMP,respectively.

In the following sections, we discuss several pieces of evi-dence which support these kinetic models.

Modeling toluene and o-xylene into the active site of ToMOA. We have tried to dock substrates into the active site cavity ofToMO A by the Monte Carlo method, as it allows effectiveexploration of the conformational space with less central pro-cessing unit time than other, more time-consuming methodssuch as molecular dynamics. In all docking experiments, thebackbone of ToMO A and the structure of the di-iron clusterwere held rigid whereas at least two layers of side chainsaround the active site cavity were allowed to move to improvethe fit of ligands inside the cavity. The Monte Carlo energyminimization of ToMO A without ligands in the active sitecavity showed that only three residues contributing to the sur-face of the cavity, i.e., Ile100, Thr201, and Phe205, were par-ticularly mobile, being able to assume several conformations.However, the mobility of Phe205 was limited to the �2 torsionangle. In the second layer of residues, Leu208, Leu272, Gln204and, to a lesser extent, His96 were able to adopt differentconformations.

When toluene and o-xylene were docked into the active site,more than 10 low-energy orientations for each substrate werefound, giving rise to several different binding complexes (datanot shown). These results suggest that aromatic substrates canassume several binding orientations inside the active site, inagreement with the models in Fig. 3.

Modeling the intermediates of benzene into the active site ofToMO A. As it is well-known that active sites are complemen-tary to activated transition states and to unstable intermediatesrather than to substrates and products (some examples can befound in references 11, 12, 24, 27, and 33 and referencestherein), we tried to identify catalytically productive bindingmodes through the docking of the intermediates of the hy-droxylation reaction. As shown in Fig. 1, two or three inter-mediates are supposed to be involved in the conversions ofaromatic hydrocarbons to phenols (16): (i) an aromatic carbo-cation, (ii) an unsaturated ketone, and possibly (iii) an epoxide.The carbocationic intermediate (CCI) has a critical role in theregioselectivity of the reaction because, after its formation, the

nature of the product is irreversibly defined. In contrast,the epoxide intermediate formed from toluene or from o-xylene can yield two different isomers, depending on whichC-O bond of the epoxide ring undergoes cleavage.

We initially tested this procedure by docking the three pos-sible intermediates of benzene hydroxylation into the activesite of wild-type ToMO. Benzene was chosen instead of tolu-ene or o-xylene because in this case a single molecular speciesexists for each intermediate. Docking was carried out by fixingthe oxygen atom transferred to the aromatic ring at the samecoordinates found for the bridging oxygen of the thioglycolateanion in the crystal structure of ToMO A (PDB code 1T0Q).The assumption that the oxygen atom is bound to the di-ironcluster even after its transfer to the substrate (Fig. 1) limits thedegrees of freedom of the intermediates and provides a fixedpoint which can be used as a rotation center for the ligand.

Binding energy values for the ToMO-benzene intermediatecomplexes reported in Table 1 indicate that the CCI fits theactive site better than the other intermediates. The main con-tribution to the tight binding of the CCI depends on electro-static interactions (Table 1), but van der Waals contacts alsoplay an important role. In the CCI, the oxygen atom is boundto a single carbon atom of the ring and the C-O bond forms anangle of �130° with the ring, which can thus be placed almostexactly in the plane of the cavity as shown in Fig. 2C, thusmaximizing the steric interaction with the cavity. On the otherhand, in the molecule of the epoxide intermediate, the six-atom ring and the epoxide ring form an angle of 105° and theoxygen atom lies above the central point of the C-C bond ofthe epoxide ring. As a consequence, when the oxygen atom islocated at the bridging position of the di-iron cluster, too-closecontacts between the six-atom ring and the active site cavityand between the three-atom ring and the di-iron cluster aregenerated (data not shown). As for the ketonic intermediate,the oxygen atom is in the same plane as the carbon atom ring.This geometry prevents the positioning of the ring in the planeof the cavity but, interestingly, pushes it toward the tunnel (Fig.2D) which connects the active site to the exterior of the mol-ecule.

An interesting observation which stems from these dockingexperiments is that the orientation of the carbocation (Fig. 2C)is very similar to that of the thioglycolate found in the crystalstructure of ToMO (this feature is even more evident in thecase of the carbocations deriving from o-xylene, as discussedbelow).

Modeling the intermediates of toluene and o-xylene into theactive site of ToMO A. Given the increased stability of the

TABLE 1. Interaction energy values for ToMO A-benzenereaction intermediates

LigandInteraction energy (kcal/mol)

van der Waals Electrostatic Total

Epoxide �12.27 �6.42 �18.69Carbocation �15.06 �19.44 �34.50Ketonea �13.41 �9.08 �22.49Ketoneb �13.35 �9.43 �22.78

a sp3 carbon near Thr201.b sp3 carbon near Ala107.



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ToMO-CCI complex relative to the ToMO-epoxide andToMO-ketone complexes, in the case of benzene we decidedto pay particular attention to the modeling of the ToMO-CCIcomplexes corresponding to toluene and o-xylene. Docking theCCIs of toluene and o-xylene is a complex procedure becauseseveral isomers exist. As shown in Fig. 4, toluene may generateup to five CCIs and four possible intermediates may be pro-duced from reactions starting with o-xylene. Intermediate cou-ples 5 and 6, 7 and 8, 10 and 11, and 12 and 13 are enantiomers.CCIs 5 and 6 yield o-cresol, CCIs 7 and 8 yield m-cresol, CCIs10 and 11 yield 2,3-DMP, and CCIs 12 and 13 yield 3,4-DMP.CCI 9 yields p-cresol.

Our docking experiments indicate that only CCIs 5, 7, and 9from toluene and CCIs 10 and 12 from o-xylene can bind to theactive site of wild-type ToMO with the same orientation as theCCI derived from benzene (data not shown), i.e., with the ringin the plane of the grids in Fig. 2B and C. As described in thefollowing sections, using only the binding energy values relative

to these five intermediates, we obtained very good agreementbetween predicted and experimentally determined percentages;therefore, we will discuss only the docking analyses of theseintermediates.

Figure 5A shows a model of the positioning of CCIs 10 and12, which lead to 2,3-DMP and 3,4-DMP, respectively, into theactive site. The models of CCIs 5 and 7, leading to o- andm-cresol, respectively, are completely superimposable onto themodel of CCI 10 (data not shown), whereas CCI 9, which yieldsp-cresol, has an orientation similar to that of CCI 12 (data notshown). Figure 5B shows that the ring of CCI 12 is placedexactly in the plane of the grid in Fig. 2 and that it mimics theorientation of thioglycolate even better than the CCI of ben-zene shown in Fig. 2C. Thus, our data indicate that CCIs 5, 7,9, 10, and 12 dock into the active site of ToMO A and placetheir methyl groups into subsites located on the border of thepocket, as hypothesized previously (4). However, the ToMO-CCI complexes suggest rather different positioning of the sub-sites for methyl groups from that in the original model. Themodel in Fig. 5A shows that only the ortho and para subsitesare unambiguously defined. In this new model, the ortho sub-site, located among residues Glu134, Leu192, and Ala107, iscloser to the di-iron cluster than that in our previous modelwhereas the new para subsite is defined by residues Glu103,Phe176, and Ile100 (the meta subsite in the original model).Moreover, two alternative meta subsites (designated m1a andm1b) can be mapped. The existence of two alternative metasubsites may depend on the close proximity of the ortho andpara subsites. This geometry is incompatible with the simulta-neous docking of three adjacent methyl groups. Therefore,when CCI 10 is docked into the cavity, the ortho methyl groupsblock the ortho subsite whereas the meta methyl group partiallyfills the para subsite, thus defining the m1b subsite (residuesGlu103, Phe176, and Met180). On the other hand, when CCI12 is docked into the cavity, the para methyl group fills the parasubsite, whereas the meta group partly occupies the ortho sub-site, thus defining the m1a subsite (residues Ala107, Glu103,and Met180). The van der Waals contributions of the methylgroups of CCI 10 (subsites o and m1b) are �1.12 and �2.02kcal/mol, respectively, whereas their contributions in the caseof CCI 12 (subsites m1a and p) are �1.64 and �1.65 kcal/mol,respectively. Hence, it seems that a methyl group positionedinto the m1b subsite gives a greater contribution to the bindingenergy than one positioned into the m1a subsite. An indirectconfirmation of this observation comes from the docking ofCCI 7, which leads to m-cresol. In this case, the single methylgroup is predicted to occupy subsite m1b.

In order to further analyze the subsites of the active site, wedocked the intermediate of the hydroxylation of m-xylene,which yields 2,4-DMP, into the cavity (Fig. 5C). In this case,both the ortho and para methyl groups were forced slightly outfrom the ortho and para subsites. Because of the steric hin-drance between the ortho methyl and residue Glu134 and be-tween the para methyl and residue Ile100, the contributions ofthe ortho and para methyl groups to the binding energy de-creased to �0.05 and �0.63 kcal/mol, respectively. These twovalues are significantly lower than those found for the inter-mediates deriving from o-xylene (see above). Interestingly, thetotal van der Waals contributions of CCIs 10 and 12 and of theintermediate produced from m-xylene to the binding energy

FIG. 4. Chemical structures of the possible CCIs deriving fromtoluene and o-xylene. CCIs 5 and 6, CCIs 7 and 8, and CCI 9 are thepossible intermediates for the transformation of toluene into o-, m-,and p-cresols, respectively. CCIs 10 and 11 and CCIs 12 and 13 are thepossible intermediates for the transformation of o-xylene into 2,3- and3,4-DMPs, respectively.



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were �15.54, �16.01, and �13.37 kcal/mol, respectively. Thisfinding would indicate that the cavity is better tailored to ac-commodate the intermediates from the physiological substrateo-xylene than those deriving from a nonphysiological substratesuch as m-xylene.

In conclusion, our docking data provide a very detailedmap of the active site residues potentially involved in regio-selectivity.

From binding energies to percentages of products. In thehypothesis that the CCI is the first intermediate formed duringthe hydroxylation reaction, as the toluene3CCI reaction isendergonic, the ET‡ transition states should be similar to en-zyme-CCI complexes. Therefore, the �nbE‡

o-m, �nbE‡m-p, and

�nbE‡o-p values defined by equations 4 to 6 can be estimated

through the Monte Carlo docking of the CCIs. Using equa-tions 1 to 6 and the binding energy values provided by theZMM software for toluene CCIs 5, 7, and 9, we predicted thepercentages of cresols. Predicted percentages of o-, m-, andp-cresols (39.7, 19.9, and 40.4%, respectively) were very similarto experimentally determined percentages (36, 19, and 45%,respectively). Similarly, using the binding energy values foro-xylene CCIs 10 and 12, we found that predicted percentagesof 2,3-, and 3,4-DMPs (17.9 and 82.1%, respectively) were verysimilar to experimentally determined percentages (19 and81%, respectively). Thus, it may be concluded that the hypoth-eses of completely steric control of regioselectivity and the useof the ToMO A-CCI noncovalent bond energies for calculatingthe relative stabilities of the ET‡ or EX‡ complexes are essen-tially correct.

As a control, the docking procedure was repeated using thetwo possible epoxides deriving from toluene, i.e., toluene 2,3-epoxide and toluene 3,4-epoxide, which provide o-cresol/m-cresol and m-cresol/p-cresol, respectively. The binding energyof toluene 3,4-epoxide was found to be about 2 kcal/mol higherthan that of toluene 2,3-epoxide. In a system at equilibrium,this energy difference would imply the formation of less than5% 2,3-epoxide. Even assuming that the 2,3-epoxide interme-diate converts entirely to o-cresol, this finding is not in agree-ment with the experimentally determined percentage ofo-cresol (36%). Similarly, in the case of the ketonic interme-diates, the isomer leading to m-cresol showed the higher bind-ing energy (data not shown), in disagreement with the exper-imental data.

Modeling the CCIs of toluene and o-xylene in the active sitesof ToMO A mutant forms. To further test our hypothesis, allthe residues located on the edge of the cavity (Ala107, Met180,Glu103, Phe176, Ile100, and Phe205) were selected for muta-tional studies to experimentally verify whether changes at thesesites would affect the regioselectivity of the enzyme in a pre-dictable way. These residues were all changed to hydrophobicresidues to preserve the hydrophobic nature of the pocket.

Ala107 was changed to larger residues, such as Val and Ile,in order to hinder the ortho site. Met180 was changed to Ile inan enzyme already carrying the mutation E103G in order toobtain a double-mutant enzyme designated (E103G, M180I)-ToMO A, in which all the residues facing the active site pocketare identical to the corresponding residues present in homol-ogous T4MO.

Glu103, previously mutated to Gly, Leu, and Met (4), waschanged to the -branched residues Val and Ile in order to

FIG. 5. Structures of the ToMO-CCI 10 and ToMO-CCI 12 com-plexes. (A) Superimposition of the ToMO-CCI 10 complex (carbonatoms are in white) onto the ToMO-CCI 12 complex (carbon atomsare in green). (B) Superimposition of the PDB structure 1T0Q (carbonatoms are in magenta) onto the structure of the ToMO-CCI 12 com-plex (carbon atoms are in green). (C) Superimposition of the com-plexes ToMO-CCI 10 and ToMO-CCI 12 (colored as in panel A) ontothe complex of ToMO with the CCI of the reaction m-xylene 32,4-DMP (carbon atoms are in magenta). The surface of the cavity ofthe complex ToMO-CCI 10 is shown as a mesh. The mesh is coloredto show the contributions of residues Glu134, Ala107, Met180, Glu103,Phe176, and Ile100.



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increase hindrance in the region between the meta and parasites. Phe176 was changed to Leu and Ile in order to enlargethis region.

Ile100 contributes to defining the hypothetical para site, butit is also at the boundary between the active site pocket and thetunnel which connects the pocket to the surface of the protein.Moreover, it is less closely packed than the other residues ofthe active site and bulges from the surface of the pocket. Thecorresponding residue in MMOs, Leu110, has been definedpreviously as the gate that controls the access to the active site(2, 29). Therefore, to explore the entire range of side chaindimensions, Ile100 was changed to Ala, Val, Leu, Met, Phe,and Trp.

Phe205, which is located at the boundary between the activesite pocket and the tunnel, like Ile100, was changed to Leu.

All mutant enzymes were assayed with phenol, benzene,toluene, o-xylene, and naphthalene. The majority of the mu-tations did not change the catalytic efficiency with respect tothat of wild-type ToMO or caused only minor changes. Onlymutations I100A, I100W, F205L, A107V, and A107I werefound to reduce significantly the kcat values for all the sub-strates (Table 2). Possible explanations are discussed briefly inthe supplemental material.

The docking procedure for the prediction of the regioselec-tivity was repeated using the Monte Carlo-optimized models ofToMO A mutant enzymes. As in wild-type ToMO A, thebackbone and the structure of the di-iron cluster were heldrigid; i.e., we assumed that the mutations did not significantlychange the structure of the protein. Recently, Murray andcoworkers have published the crystallographic structure ofToMO carrying the mutation I100W [the (I100W)-ToMO en-zyme] (18). In spite of the large increase in the hindrance ofthe side chain at position 100, the structure of the mutatedToMO A is essentially unchanged. This effect happens becausethe bulky tryptophan side chain is accommodated inside theactive site cavity partially hindering it. As all the residues wehave mutated are at the border of the active site cavity, it islikely that variations in the steric hindrance of these side chainscan be easily accommodated without relevant changes to thebackbone structure, as observed in the case of the I100Wmutation. We want also to underline that the Monte Carlo-optimized model of (I100W)-ToMO correctly predicted theorientation of the mutated side chain (data not shown).

Tables 3 and 4 report the results obtained after the dockingof toluene- and o-xylene-derived CCIs, respectively, to ToMOA mutant enzymes at positions 107, 103, 180, and 176. Theregioselectivities of all mutant enzymes, with the exception ofthose of (E103L)-ToMO for toluene and (F176L)-ToMO foro-xylene, were predicted with fairly good accuracy. Minor dif-ferences between experimental and predicted percentages maydepend on the small differences in binding energy values (�G‡)

TABLE 2. Apparent kcat values of ToMO and ToMO mutantproteins on benzene, toluene, o-xylene, and naphthalene

ToMO variantor mutation(s)

kcat (s�1)a for substrate:

Phenol Benzene Toluene o-Xylene Naphthalene

Wild type 1.0 0.37 0.43 0.26 0.033I100A 0.019 0.05 0.03 0.025 0.0015I100V 0.4 0.22 0.27 0.19 0.011I100M 0.35 0.14 0.18 0.078I100L 0.76 0.3 0.56 0.3I100F 0.15 0.13 0.3 0.16I100W 0 0.018 Very low Very lowF176I 0.25 0.43 0.28 0.21 0.016F176L 0.32 0.36 0.32 0.23 0.016A107V 0.07 0.064 0.024 0.032A107I 0.027 0.0017 0.0009 0.0005F205L 0.01 0.0015 0.00094 0.0006E103G 1.0 0.43 0.42 0.42E103L 0.89 0.29 0.32 0.36E103 M 0.29 0.26 0.3 0.2E103V 0.24 0.22 0.22 0.11E103I 0.21 0.03 0.03 0.01E103G, I100L 0.53 0.58 0.67 0.75E103V, I100V 0.30 0.11 0.097 0.029E103G, M180I 0.50 0.22 0.34 0.2

a Standard errors for kcat values were �20% for phenol and �25% for ben-zene, toluene, o-xylene, and naphthalene.

TABLE 3. Comparison between experimentally determined and calculated percentages of cresol isomers produced by wild-type ToMO andToMO mutant proteins

ToMO variant ormutation(s)

Binding energya (kcal/mol) for: Calculated % ofb: Experimentallydetermined %c of:

ET‡o ET‡

m1b ET‡m1a ET‡

p o-C m-C p-C o-C m-C p-C

Wild type �34.69 �34.28 �34.70 39.7 19.9 40.4 36 19 45E103G �34.00 �33.91 �35.44 7.5 6.5 86.0 9 6 85E103G, M180I �32.74 �33.85 �35.28 1.2 8.1 90.7 6 10 84A107V �27.50 NAd NA �34.04 0.0 2.9 97.1 0 6 94A107I �27.30 NA NA �33.90 0.0 3.3 96.7 0 4 96F176I �33.28 �33.31 �34.91 5.6 5.9 88.4 7 6.5 87.5F176L �33.27 �33.40 �34.82 6.3 7.8 85.9 6 7 87E103V �33.70 �33.35 �33.45 45.2 25.1 29.7 49 34 17E103I �33.73 �33.03 �32.89 64.6 19.8 15.6 59 24 17E103M �33.86 �33.29 �33.21 58.5 22.3 19.2 47 19 34E103L �33.46 �31.61 �32.26 85.1 3.7 11.2 20 11 69

a Total binding energy for the complex ToMO variant-CCI 5 (ET‡o), ToMO variant-CCI 7 (ET‡

m1b and ET‡m1a), or ToMO variant-CCI 9 (ET‡

p).b o-, m-, and p-C, o-, m-, and p-cresols.c Error, �1%.d NA, not applicable. The hindrance of the branched side chains of Val and Ile forces the meta methyl group to occupy a position intermediate between the m1b

and p subsites. The binding energies for the (A107V)-ToMO–CCI 7 and (A107I)-ToMO–CCI 7 complexes are �31.95 and �31.90 kcal/mol, respectively.



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among ET‡o, ET‡

m, ET‡p, EX‡

3,4, and EX‡2,3 complexes. In

fact, our approach predicts �G‡ values of 0.1 to 0.3 kcal/mol,which are 1% or less of the calculated binding energy values(30 to 36 kcal/mol).

Moreover, as the docking procedure provides the bindingenergy values of each EI, it is now possible to understand alsohow each mutation influences the stability of the interaction oftoluene and o-xylene methyl groups at each subsite. For exam-ple, mutation E103G significantly reduces the production ofo-cresol, increasing the difference in stability between the orthoand para orientations, which changes from 0.01 kcal/mol in thecase of the wild-type protein to 1.44 kcal/mol in the case of theE103G mutant enzyme. As shown in Table 3, this increase isdue both to the destabilization of the ortho orientation (com-plex ET‡

o) and to the stabilization of the para orientation(complex ET‡

p). Moreover, the ZMM software yields individ-ual group contributions to the total binding energy (see Ma-terials and Methods). Our data (not shown) indicate that thecontribution of the methyl group in the ortho site to the bindingenergy, due essentially to van der Waals interactions, is barelyinfluenced by the E3G mutation, changing from �1.34 kcal/mol for the wild-type protein to �1.47 kcal/mol for the E103Gmutant form. Rather, the predicted destabilization of the ET‡

o

complex (0.69 kcal/mol) depends on the loss of van der Waalscontacts between the C-H groups at positions 3 and 4 of thering (Fig. 4 numbering) and the larger active site pocket. Onthe other hand, the enlargement of the para subsite removesclashes between the para methyl group and the pocket andincreases the contribution of the para methyl group to thebinding energy (from �0.76 to �2.1 kcal/mol). In the case ofo-xylene, the mutation E103G increases the relative stability ofthe EX‡

3,4 complex, which provides 3,4-DMP, both increasingthe stability of the EX‡

3,4 complex and decreasing the stabilityof the EX‡

2,3 complex (Table 4). The values for the individual

components of the calculated binding energy indicate that theincrease in the stability of the EX‡

3,4 complex is due mainly toimproved binding of the methyl groups and that the decreasein the stability of the EX‡

2,3 complex depends on the worseaccommodation of the substrate ring.

Mutations F176I and F176L have effects similar to that ofmutation E103G on the regioselectivity of ToMO A, but theseeffects depend on different factors. Indeed, these two muta-tions leave the stability of the ET‡

p complex (Table 3) almostunchanged with respect to that of the wild-type enzyme,whereas they decrease the stability of ET‡

o and ET‡m com-

plexes by 1.42 and 0.95 kcal/mol, respectively. Moreover, thevalues for the individual components of the binding energyindicate that the decrease in the stability of the ET‡

o complexis due mainly to a loss of van der Waals contacts between theactive site pocket and the ring of the substrate (about 0.9kcal/mol) and that the contribution of the methyl group to thebinding energy is not affected by the mutation (the change isless than 0.1 kcal/mol). In contrast, in the case of the ET‡

p

complex, the loss of van der Waals contacts between the activesite pocket and the ring of the substrate (about 0.8 kcal/mol) iscounterbalanced by an increased contribution by the methylgroup of the substrate (about 1 kcal/mol).

As for mutations E103I and E103V, it should be noted thatthey insert -branched residues at position 103. This insertiongenerates clashes between the side chain and the ring of thesubstrates, thus lowering the stability of all the ToMO-CCIcomplexes with respect to that of the wild-type enzyme (Tables3 and 4). The greater destabilization of the ET‡

p complex thanof ET‡

o and ET‡m (Table 3) leads to increased production of

o- and m-cresols, whereas slightly different conformations ofthe valine and isoleucine side chains (data not shown) makethe (E103V)-ToMO–ET‡

m1a complex more stable than the(E103I)-ToMO–ET‡

m1b complex. These results give a molec-

TABLE 4. Comparison between experimentally determined and calculated percentages of DMP isomers produced by wild-type ToMO andToMO mutant proteins


Binding energya (kcal/mol) for: Calculated % of: Experimentallydetermined %b of:

EX‡2,3 EX‡

3,4 2,3-DMP 3,4-DMP 2,3-DMP 3,4-DMP

Wild type �35.27 �36.17 17.9 82.1 19 81E103G �35.18 �36.39 11.5 88.5 1 99E103G, M180I �34.1 �36.23 2.7 97.3 2 98A107V �26.20 �32.65 0.0 100 0 100A107I �25.60 �31.80 0.0 100 0 100F176I �33.60 �35.25 5.8 94.2 2.5 97.5F176L �34.8 �35.47 24.4 75.6 2.5 97.5E103V �33.81 �34.15 36.0 64.0 39 61E103I �31.67 �33.20 7.0 93.0 13 87E103M �34.36 �35.02 24.7 75.3 18 82E103L �32.75 �33.76 15.4 84.6 6 94

I100A �34.61 �35.85 11.0 89.0 18 82I100V �35.33 �36.01 24.1 75.9 12 88I100L �35.43 �36.16 22.6 77.4 12 82I100M �35.48 �36.51 15.0 85.0 28 72I100F �35.79 �36.84 14.5 85.5 19 81I100L, E103G �35.09 �36.29 11.6 88.4 4 96I100V, E103V �33.62 �34.17 28.3 71.7 34.5 65.5

a Total binding energy for the complex ToMO variant-CCI 10 (EX‡2,3) or ToMO variant-CCI 12 (EX‡

3,4).b Error, �1%.



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ular reason for the enhanced production of m-cresol by mutant(E103V)-ToMO with respect to that by mutant (E103I)-ToMO. Moreover, due to the slightly different orientations ofthe two side chains, mutation E103V decreases the stability ofthe EX‡

3,4 complex much more than that of the EX‡2,3 com-

plex and mutation E103I has the opposite effect (Table 4). Asa consequence, mutation E103V increases the percentage of2,3-DMP whereas mutation E103I decreases it with respect tothat produced by wild-type ToMO. In both cases, the decreasein the stability of the EX‡

2,3 and EX‡3,4 complexes is due to

worse accommodation of both the substrate ring and themethyl groups.

The Ala107 side chain, according to our model, contributesto the surface of the ortho site. In agreement with the model,the mutation of Ala107 to Val or Ile, whose side chains fill thecavity of the ortho site, completely abolishes the production ofo-cresol and 2,3-DMP.

Modeling intermediates in the case of Ile100 mutant en-zymes. Several mutations at position 100, which defines thepara subsite, surprisingly increase the production of m-cresol(Table 5). Replacement with alanine, leucine, phenylalanine,and valine increases the production of m-cresol, whereas re-placement with methionine and tryptophan decreases it. It isalso interesting that the effects of mutations at positions 100and 103 are additive, as shown by the behavior of the double-mutant enzymes (I100L, E103G)-ToMO A and (I100V,E103V)-ToMO A. The individual mutations I100V and E103Vincrease the percentage of m-cresol from 19 to 47 and 34%,respectively, whereas the corresponding double mutant pro-duces 62% m-cresol. Furthermore, mutation I100L increasesthe percentage of m-cresol from 19 to 38%, whereas mutationE103G decreases it to 6%. In this case, the double-mutantenzyme (I100L, E103G)-ToMO A produces 17% m-cresol. Asshown in Fig. 5A, a second hypothetical meta site (the m2 site)may exist between Ile100 and Phe205 (the para subsite in ourprevious model [4]) which is occupied by the methyl groupwhen CCI 8 (Fig. 4) is docked into the active site. However, thebinding of CCI 8 is different from the binding of the otherintermediates because the hindrance of the side chains ofIle100 and Phe205 forces the ring of the intermediate into aplane which forms an angle of about 25° with the plane that

contains intermediates 5, 7, 9, 10, and 12 (Fig. 6A). Thus, itmay be that this different geometry of the wild-type ToMOA-CCI 8 complex—with the aromatic ring out of the plane ofthe grid in Fig. 2B—makes the complex catalytically unproduc-tive. In fact, this geometrical constraint is the reason that, inthe case of the wild-type protein, we have limited our dockinganalysis to intermediates 5, 7, 9, 10, and 12 only (see above).

Several mutations of residue 100 remove part or all of thehindrance and allow intermediate 8 to dock to the active site ina conformation more similar to those of intermediates 5, 7, 9,10, and 12 (Fig. 6A). This finding makes kinetic model i inad-equate to predict the effects of mutations at position 100. Totake into account the extra methyl subsite m2, we modifiedmodel i by introducing a fourth ET complex (ETm2) and thecorresponding transition state ET‡

m2 (model iii in Fig. 3).Model iii includes three extra �G‡ values, �G‡

o-m2, �G‡m1-m2,

and �G‡p-m2, which determine the relative abundance of the

ET‡m2 complex with respect to total ET‡ complexes and,

hence, the amount of m-cresol produced by the pathway ETm2

^ ET‡m2 3 enzyme-CCI 8.

As the orientation of CCI 8 inside the active site is differentfor each mutant protein and different from those of interme-diates 5, 7, 9, 10, and 12, the hypothesis that the �G‡ valuesdepend essentially on the noncovalent bond energy contribu-tions is no longer valid. On the contrary, because of nonopti-mal binding of the activated substrate to the cluster, the cova-lent bond energy component of the ET‡

m2 complex, bE‡m2,

should be generally higher than that of the other three ET‡

complexes, bE‡:

�G‡o-m2 � ��nbE‡

m2 � nbE‡o� � �bE‡

m2 � bE‡�� nbE‡m2-o

� �bE‡� (7)

�G‡m1-m2 � ��nbE‡

m2 � nbE‡m1� � �bE‡

m2 � bE‡�� nbE‡m2-m1

� �bE‡� (8)

�G‡p-m2 � ��nbE‡

m2 � nbE‡p� � �bE‡

m2 � bE‡�� nbE‡m2-p

� �bE‡� (9)

where �bE‡ (bE‡m2 � bE‡) � 0.

TABLE 5. Comparison between experimentally determined and calculated percentages of cresol isomers produced by wild-type ToMO andToMO variants mutated at position 100


Binding energy (kcal/mol)a for:e��bE‡/RT

Calculated % ofb: Experimentallydetermined %c of:

ET‡o ET‡

m1b ET‡m1a ET‡

p ET‡m2 o-C m-C p-C o-C m-C p-C

Wild type �34.69 �34.28 �34.70 �34.50 0.00 40.0 20.0 40.0 36 19 45I100A �33.90 �33.88 �35.07 �35.20 0.58 7.0 43.0 50.0 9 42 49I100V �33.80 �33.86 �34.30 �34.75 0.38 16.0 47.0 37.0 13 47 40I100L �34.66 �34.15 �34.35 �35.23 0.32 35.0 44.0 21.0 31 38 31I100 M �34.48 �33.98 �35.04 �37.76 0.00 22.0 10.0 68.0 29 5 66I100F �34.71 �34.52 �35.85 �36.02 0.90 5.0 52.0 42.0 37 35 28I100W �34.55 �34.40 �36.52 �36.29 0.55 3.0 28.0 70.0 25 5 70I100L, E103G �33.98 �33.81 �35.30 �34.08 1.00 8.0 16.0 76.0 6 17 77I100V, E103V �35.50 �32.85 �33.46 �35.51 2.70 20.0 61.0 19.0 19.5 62 18.5

a Total binding energy for the complex ToMO variant-CCI 5 (ET‡o), ToMO variant-CCI 7 (ET‡

m1b and ET‡m1a), ToMO variant-CCI 9 (ET‡

p), or ToMO variant-CCI8 (ET‡

m2).b o-, m-, and p-C, o-, m-, and p-cresols.c Error, �1%.



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Therefore, the equilibrium constants which determine therelative abundance of the ET‡

m2 complex can be expressed asfunction of the �bE‡ values:

K‡o-m2 � �ET‡

m2�/�ET‡o� � exp� � �nbE‡

m2-o/RT�

� exp� � �bE‡/RT� (10)

K‡m1-m2 � �ET‡

m2�/�ET‡m1� � exp� � �nbE‡

m2-m1/RT�

� exp� � �bE‡/RT� (11)

K‡p-m2 � �ET‡

m2�/�ET‡p� � exp� � �nbE‡

m2-p/RT�

� exp� � �bE‡/RT� (12)

Using the Monte Carlo In equations 10 through 12, correct toadd symbol “�” between “exp . . .” expressions [e.g.,exp(��nbE‡m2-p/RT) � exp(��bE‡/RT)]? If not, please clar-ify relationship between these expressions; should space be-

tween them be deleted, or should a chem point or some othersymbol be added in place of the multiplication sign?� strategy,we predicted nbE‡

o, nbE‡m1, nbE‡

p, and nbE‡m2 for each muta-

tion at position 100. Then, using equations 1 to 12, we deter-mined the exp(��bE‡/RT) values, which provided predictedcresol percentages similar to the experimentally determinedones. Table 5 shows that, except for mutations I100F andI100W, for each mutation at position 100, a single exp(��bE‡/RT) value exists which yields good agreement between exper-imental and predicted percentages of products. Moreover, Fig.6B shows that there is good correlation between theseexp(��bE‡/RT) values and the angle � formed between theplane in which intermediates 5, 7, 9, 10, and 12 lie (the planeof the grid in Fig. 2) and the plane where intermediate 8 islocated (Fig. 6A). This interesting finding suggests that theenergy of the transition state increases with the angle � andfurther supports the hypothesis that the predicted MonteCarlo-minimized complexes ToMO A-benzene-CCI andToMO A-CCI 5, 7, 9, 10 or 12 illustrate the geometry of thecatalytically productive orientation of aromatic substrates in-side the ToMO A active site pocket. Moreover, we want also tounderline that our model provides a simple explanation for theadditive effects of mutations at positions 100 and 103. In fact,according to our model, the observed percentage of m-cresol isthe sum of the percentages of m-cresol produced from toluenedocked with its methyl group in subsite m1 and from toluenedocked with its methyl group in subsite m2. Moreover, theresidue at position 103 prevalently contributes to the m1 site,whereas the residue at position 100 contributes to the m2 site.Thus, the combined effect of a mutation at position 103, whichimproves interactions at subsite m1, and a mutation at position100, which partially opens subsite m2, is the production of apercentage of m-cresol close to the sum of the percentagesproduced by the single mutations. This is the case for thedouble mutant (I100V, E103V)-ToMO. In the case of thedouble mutant (I100L, E103G)-ToMO, the effect of mutationE103G, which decreases the ability of the m1 site to anchor amethyl group, is counterbalanced by the effect of mutationI100L on the catalytic efficiency of the m2 site. Consequently,no apparent change in m-cresol production is observed.

As for mutations I100F and I100W, the lower level of agree-ment between predicted and experimental data may be due tothe fact that mutations I100F and I100W increase significantlythe volume of the side chain at position 100. As describedabove, the crystallographic structure of the (I100W)-ToMOmutant enzyme (18) shows that the tryptophan side chainpoints toward the active site cavity, partially hindering it. Mostlikely, the simultaneous accommodation inside the active sitecavity of the bulky side chain of tryptophan—or phenylala-nine—and of the substrate may require changes in the confor-mation of the ToMO A backbone which cannot be predictedby our strategy, as all the docking experiments were carried outby holding the backbone of ToMO A in a rigid position andallowing the movement of only the side chains closer to thecavity.

As for o-xylene, our docking data indicate that there is nomutation at position 100 which allows the positioning of inter-mediates 11 and 13 in the active site with a conformationsimilar to those of intermediates 10 and 12 (data not shown).Thus, in the case of o-xylene, there is no need for hypothesizing

FIG. 6. Comparison between the orientation of CCI 10 docked intowild-type ToMO (WT) and the orientations of CCI 8 docked intowild-type and mutant ToMOs. (A) The surface of the cavity of theToMO-CCI 10 complex is shown as a mesh. � is the angle between thering of CCI 10 docked into wild-type ToMO and CCI 8 docked intowild-type and mutant ToMOs. (B) Correlation between angle � shownin panel A and exp(��bE‡/RT) values. Data for the mutation I100Vwere not used in the linear fit.



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a kinetic model which takes into account the involvement of anm2 subsite. Consequently, we used kinetic model ii and calcu-lated the data reported in the lower part of Table 4. Also in thiscase, the predicted regioselectivities of the mutant enzymeswere in good agreement with the experimental values.

Regioselectivity on naphthalene. From a steric point of view,naphthalene can be described as an ortho disubstituted ben-zene derivative bearing groups larger than methyl groups. FourCCIs can be produced from naphthalene, similar to thoseshown in Fig. 4 for o-xylene. Our docking data indicate thatonly intermediates equivalent to CCIs 10, 12, and 13 in Fig. 4can fit the active site (in Table 6, these complexes are namedEN‡

�, EN‡ 1, and EN‡

2, respectively, and their models areshown in Fig. S5 in the supplemental material). However, noneof these three intermediates can assume exactly the same ori-entation as the intermediates derived from benzene, toluene,and o-xylene. This finding is in agreement with the observationthat the kcat value of wild-type ToMO for naphthalene is con-siderably lower than those measured for the more physiologi-cal substrates. Data in Table 6 show that in the case of naph-thalene, our predictions are only qualitatively correct. In fact,for all the mutant enzymes we have studied, the calculated�-naphthol/ -naphthol ratio was higher than that observedexperimentally. However, the model correctly predicts thatmutations which reduce the volume of the residues at positions100 and 176 (Ile and Phe, respectively) increase the percentageof -naphthol. Ile and Phe side chains likely impair the correctpositioning of the naphthalene reaction intermediates corre-sponding to CCIs 12 and 13 (i.e., the intermediates which formthe complexes EN‡

1 and EN‡ 2, respectively).

The poor quantitative agreement of our predictions withexperimental data may depend on the rigidity of the backboneand of the major part of the side chains in the docking proce-dure. It may be possible that greater flexibility of the backboneis needed to accommodate this large substrate. Moreover, itshould also be remembered that the models shown in Fig. 3 arebased on the hypothesis that EX complexes are generatedthrough fast equilibrium events. From a molecular point ofview, this means that substrates can easily change their orien-tation inside the active site. Most likely, the hypothesis holdstrue for monocyclic, small substrates, but it could not hold inthe case of a bulky molecule like naphthalene (see Fig. S5 inthe supplemental material). If naphthalene, entering the activesite, generates an EN complex (as the docking of naphthalene

CCIs suggests [see Fig. S5C in the supplemental material]) andthe rate of conversion of this initial complex to EN� is nothigher than the rate of the hydroxylation reaction, more -naphthol than the amount predicted by a model based onfast equilibrium events will be formed.

Regioselectivity on polar substrates: the case of phenol. Anintriguing feature of several multicomponent monooxygenasesis their specificity in the second hydroxylation step, which pro-duces exclusively catechol from phenol and (di)methyl-catechols from cresols and DMPs. It should also be remem-bered that Tao et al. and Vardar and Wood (34, 35), usingrandom mutagenesis, were able to obtain several ToMO andT4MO mutant forms which produce hydroquinone in differentamounts. Mutant (I100Q)-ToMO is particularly interesting, asit produces 80% hydroquinone and only 20% catechol, thephysiological product of ToMO. Even if a detailed analysis isbeyond the scope of this paper, we have tested our approachwith this mutant enzyme and with wild-type ToMO.

We have docked the two possible enantiomeric CCIs of thephenol-to-catechol hydroxylation reaction (CCIs 14 and 15 inFig. 7) into the ToMO active site on the hypothesis that, likethose for methyl groups, subsites for the hydroxyl group shouldexist.

Our results indicate that the hydroxyl groups can be posi-

FIG. 7. Chemical structures of the CCIs deriving from phenol.CCIs 14 and 15 are the possible intermediates of the phenol-catecholreaction. CCI 16 is the intermediate of the phenol-hydroquinone re-action. The positive charge is delocalized on both the ring and the OHgroup.

TABLE 6. Comparison between experimentally determined and calculated percentages of naphthol isomers produced by wild-type ToMOand ToMO mutant proteins

ToMOvariant ormutation

Binding energya (kcal/mol) for: Calculated %b of: Experimentallydetermined %c of:

EN‡� EN‡

1 EN‡ 2 �-N -N produced

by EN‡ 1

-N producedby EN‡

2�-N -N

Wild type �38.66 �31.08 �35.49 99.5 0.0 0.5 87 13F176I �36.32 �32.88 �34.10 97.4 0.3 2.3 57 43F176L �37.29 �28.65 �34.17 99.5 0.0 0.5 57 43I100A �38.58 �33.37 �36.10 98.5 0.02 1.5 53 47I100V �38.02 �30.30 �35.91 97.3 0.0 2.7 81 19

a Total binding energy for the complex ToMO variant-naphthalene analogue of CCI 10 (EN‡�), ToMO variant-naphthalene analogue of CCI 12 (EN‡

1), or ToMOvariant-naphthalene analogue of CCI 13 (EN‡

2).b �- and -N, �- and -naphthol.c Error, �1%.



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tioned in two hydrophilic sites defined by residues Glu134 andGlu197 on one side of the di-iron cluster (see Fig. S6A in thesupplemental material) and by Glu231 on the other side (seeFig. S6B in the supplemental material). The contributions ofthe hydrogen bonds to the binding energy are about 0.5 kcal/mol in both cases. When the intermediate leading to hydro-quinone (CCI 16 in Fig. 7) was docked into the active site ofToMO, we noticed that no hydrogen bond partner was presentin the catalytic pocket, thus giving a molecular basis for theinability of the wild-type enzyme to produce hydroquinone. Onthe contrary, in the (I100Q)-ToMO mutant enzyme, the car-bonyl group of the Gln100 side chain could form a hydrogenbond with the hydroxyl group on the intermediate, leading tohydroquinone (see Fig. S6C in the supplemental material),with an estimated contribution to the binding energy of 0.7kcal/mol. This finding led to a prediction which is in agreementwith the experimental observation that the (I100Q)-ToMOmutant enzyme produces more hydroquinone than catechol.

Conclusions. BMMs have broad substrate specificities, cou-pled with specific regioselectivity properties, in the hydroxyla-tion reaction of aromatic substrates. These features are meta-bolically relevant, because they are the basis for the capabilitiesof several microorganisms to grow on selected molecules.Moreover, given the catalytic potentials of BMMs, they mayconstitute a powerful tool for the bioremediation of harmfulsubstances and may serve as specific biocatalysts in (regio)s-elective syntheses.

Results from several structural and functional studies sug-gest that the different regioselectivities of BMMs depend ondifferences in the shape of the active site pocket (2, 4, 7, 17, 26,30, 34, 35). However, a detailed description of the molecularbasis for the regioselectivities of these enzymes is still lacking.This situation is particularly inconvenient because it impairsthe possibility to attempt rational modifications to producenew catalysts and/or new microorganisms endowed with spe-cific, advantageous properties.

In this study, we have developed a procedure based on thedocking of the intermediates of the hydroxylation reaction intothe active site pocket of a specific monooxygenase, ToMO A.This approach allows for (i) a detailed analysis of the molec-ular determinants of the enzyme’s regioselectivity, (ii) the pre-diction of the regioselectivity properties of mutant forms of theenzyme, in the absence of any experimental data, and (iii) theprediction of the catalytically productive orientation of a sub-strate inside the active site pocket. Thus, this procedure is avaluable tool for the design of mutant monooxygenases for usein biosynthesis and bioremediation procedures, and its appli-cability may also be extended to other kinds of substrates andother multicomponent monooxygenases.

Finally, the results of the docking experiments reported inthis paper have very interesting implications for the catalyticmechanism of TMOs. The optimal fit between the ToMOactive site pocket and the delocalized carbocation and the goodagreement between experimentally determined regioselectivityand the regioselectivity predicted using the delocalized carbo-cations as ligands strongly suggest that the delocalized carbo-cation is a crucial intermediate in aromatic hydroxylation re-actions.

ACKNOWLEDGMENTS

We are indebted to Giuseppe D’Alessio, Matthew H. Sazinsky, andAnna Tramontano for critically reading the manuscript.

This work was supported by grants from the Ministry of Universityand Research (PRIN/2002 and PRIN/2004).

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Molecular Determinants of the Regioselectivity of Toluene/o … · Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli Federico II, Complesso Universitario di