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Applied and Environmental Microbiology, October 2007, p. 6460-6467, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00823-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutagenesis of the "Leucine Gate" To Explore the Basis of Catalytic Versatility in Soluble Methane Monooxygenase{triangledown}

Elena Borodina,1,{dagger} Tim Nichol,2,{dagger} Marc G. Dumont,1 Thomas J. Smith,2* and J. Colin Murrell1

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom,1 Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom2

Received 11 April 2007/ Accepted 7 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent nonheme oxygenase that naturally oxidizes methane to methanol and can also cooxidize a wide range of adventitious substrates, including mono- and diaromatic hydrocarbons. Leucine 110, at the mouth of the active site in the {alpha} subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control the access of substrates to the active site. Previous crystallography of the wild-type sMMO has indicated at least two conformations of the enzyme that have the "leucine gate" open to different extents, and mutagenesis of homologous enzymes has indicated a role for this residue in the control of substrate range and regioselectivity with aromatic substrates. By further refinement of the system for homologous expression of sMMO that we developed previously, we have been able to prepare a range of site-directed mutations at position 110 in the {alpha} subunit of sMMO. All the mutants (with Gly, Cys, Arg, and Tyr, respectively, at this position) showed relaxations of regioselectivity compared to the wild type with monoaromatic substrates and biphenyl, including the appearance of new products arising from hydroxylation at the 2- and 3- positions on the benzene ring. Mutants with the larger Arg and Trp residues at position 110 also showed shifts in regioselectivity during naphthalene hydroxylation from the 2- to the 1- position. No evidence that mutagenesis of Leu 110 could allow very large substrates to enter the active site was found, however, since the mutants (like the wild type) were inactive toward the triaromatic hydrocarbons anthracene and phenanthrene. Thus, our results indicate that the "leucine gate" in sMMO is more important in controlling the precision of regioselectivity than the sizes of substrates that can enter the active site.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soluble methane monooxygenase (sMMO) is one of two enzyme systems via which methane-oxidizing bacteria catalyze the oxygenation of methane to methanol, which is the particularly challenging first step in the metabolism of the kinetically unreactive methane molecule (28). sMMO is a multicomponent enzyme encoded by the six-gene operon mmoXYBZDC, in which the active binuclear iron center (8, 45) that is the site of methane oxidation resides within the {alpha} subunit of the hydroxylase component, encoded by mmoX (5). It may be because methane is a small and unfunctionalized substrate that the hydrophobic pocket on the hydroxylase component of sMMO that has evolved to bind methane is also able to accommodate a very wide range of hydrocarbons and other molecules. Indeed, sMMO, whose known substrates number over 100, range in size from methane to naphthalene (2) and biphenyl (23), and also include carbon monoxide and ammonia (37), must surely be among the most catalytically versatile of all known enzymes.

The unusual catalytic versatility of sMMO has led to interest in its potential as a biocatalyst for bioremediation and synthetic chemistry as well as an interest in how the structure of the enzyme facilitates so wide a substrate range. The hydrophobic substrate binding site of sMMO, which is believed to be responsible for binding its wide range of substrates, is adjacent to the active binuclear iron center and is deeply buried in the 250-kDa ({alpha}ß{gamma})2 hydroxylase component (9, 12, 31), presumably to prevent the solvent from quenching the highly oxidizing diferryl intermediate Q (22, 33, 35) that is needed to oxygenate methane and other recalcitrant substrates. Access to the active site is likely to be via another hydrophobic pocket, cavity two, which is part of a chain of buried cavities that communicate between the active site and the solvent. Between cavity two and the active site lies the "leucine gate," residue Leu 110. Different crystal forms of the hydroxylase differ in the conformation of Leu 110, such that in the crystal state it can either block the pathway between cavity two and the active site or (in the alternative conformation) open a 2.6-Å-diameter channel between the two cavities. A larger conformational change, such as may be caused by interaction with the other components of the sMMO complex, could open this "leucine gate" further to allow passage of substrates and products (30), reasonably acting to control the access of substrates to the active site.

Other monooxygenases homologous to sMMO, which naturally oxidize molecules considerably larger than methane, have narrower substrate ranges than sMMO (21). There is an X-ray crystal structure for one of these enzymes, toluene o-xylene monooxygenase, in which the opening to the active site is wider than that observed in sMMO (33). While a more open active site is consistent with the larger natural substrate of this enzyme, it is clear that the active site of sMMO can open sufficiently to allow entry of aromatic substrates.

Mutagenesis studies of homologous monooxygenases whose natural substrates are monoaromatic hydrocarbons have indicated that the position equivalent to Leu 110 is indeed important in the interaction between these enzymes and their substrates. In a directed evolution study of toluene 2-monooxygenase of Burkholderia cepacia G4 (4), a Val-to-Ala mutation at position 106, equivalent to Leu 110 in sMMO, gave increased naphthalene and phenanthrene oxidation but no change in regioselectivity with naphthalene. Both the mutant and the wild type gave 1-naphthol as the predominant product. This was consistent with the proposed gating role of this residue, i.e., the mutation affected the rates of oxidation of large substrates but did not greatly affect regioselectivity. Similar results were found in a saturation mutagenesis study of toluene 4-monooxygenase, where the V100L mutant showed a twofold increase in the rate of oxidation of 2-methoxyphenol; here, a modest loss of regiospecificity was observed, but the mutant (like the wild type) gave 3-methoxyresorcinol as the major product (41). A reduction in the rate of oxindole formation from indole by the Ala, Cys, and Val mutations of the equivalent Ile 100 residue in toluene 4-monooxygenase (40) was also consistent with such a function, although a change in the predominant product of phenol hydroxylation from catechol to hydroquinone (i.e., a shift in regioselectivity from the 2- to the 4- position) in the I100Q mutant of toluene o-xylene monooxygenase (42) indicates that the site equivalent to Leu 110 may have a role in determining regioselectivity as well as substrate access. Results from mutagenesis of the nearby site Gly 113 in soluble butane monooxygenase have also suggested that a conformational change involving the "leucine gate" (Leu 109 in this enzyme) may be necessary for product release from the active site, since the G113N mutant showed a reduced inhibition by nascent methanol produced by oxidation of methane (13).

In order to probe the role of Leu 110 in sMMO, we here report further refinements in the homologous expression system for sMMO that we previously developed (25, 38) in order to overcome problems associated with expression of the enzyme in Escherichia coli. Several mutations at position 110 in the {alpha} subunit of the hydroxylase have been analyzed using bulky aromatic substrates in order to evaluate the role of this residue in the sMMO system and to compare its function to that of the equivalent residues in the aromatic monooxygenases.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, plasmids, and culture conditions.
Details of plasmids and methanotroph strains used during this study are given in Table 1. Plasmid construction and site-directed mutagenesis were accomplished using E. coli strains INV{alpha}F', TOP10 (Invitrogen), and XL-10 GOLD (Stratagene). The donor strain for conjugation of plasmids into Methylosinus trichosporium was E. coli S17-1 (36). E. coli strains were cultivated at 37°C in LB medium or on LB agar plates. Strains of M. trichosporium were cultivated at 30°C on nitrate mineral salts (NMS) agar plates and in NMS medium in flask and fermentor culture, using methane as the carbon and energy source, as described previously (38). As previously described, cells were cultivated in a fermentor in NMS medium containing 0.1 mg liter–1 (0.4 µM) of CuSO4·5H2O and induction of sMMO occurred when the culture optical density at 600 nm (OD600) exceeded about 5.0, when the copper-to-biomass ratio became low enough to induce sMMO genes. Where necessary, antibiotics were added to culture media at the following working concentrations: ampicillin, 50 to 100 µg ml–1; gentamicin, 5 µg ml–1; kanamycin, 50 µg ml–1.


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  		TABLE 1. Plasmids and M. trichosporium strains

 
General DNA methods.
General methods for DNA purification and analysis and cloning with E. coli were performed according to Sambrook et al. (32). Where necessary, to facilitate difficult plasmid manipulations, commercially prepared competent E. coli cells (TOP10 [Invitrogen] or Solo Pack GOLD [Stratagene]) were used. DNA probes were labeled by random priming (10, 11) using hexanucleotide primers and deoxynucleoside triphosphates from Roche according to the manufacturer's instructions. An agarose gel-purified DNA fragment (25 to 50 ng) was labeled with [{alpha}-32P]dGTP (50 µCi) for 1 h at 37°C with Klenow polymerase (Invitrogen). The unincorporated label was removed using MicroSpin columns (Amersham Pharmacia Biotech). The probes were denatured by the addition of NaOH to a final concentration of 0.4 M and incubated for 2 min at room temperature before being added to the hybridization solution.

Construction of plasmids.
In order to allow subsequent manipulations of the sMMO operon by using BamHI, the BamHI site was deleted from the vector pBBR1MCS-5 (20) by removing the fragment of the multiple cloning site that contained it with ClaI and BstBI and religating the plasmid backbone via the compatible cohesive ends thus produced, giving the vector pBBR1MCS-5b. The 10-kb fragment containing the mmoXYBZDC operon from M. trichosporium OB3b and flanking sequences was excised from pTJS176 (38) with KpnI and cloned into the unique KpnI site of pBBR1MCS-5b, giving plasmid pMD2. To direct deletion of the chromosomal copy of the sMMO-encoding operon, the plasmid pMD-Mdel3 was then constructed as detailed in Fig. 1.


Figure 1
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  		FIG. 1. Construction of the plasmid pMD-Mdel3 and its use for marker exchange deletion of the first five genes of the sMMO operon. In addition to the sMMO-encoding mmoXYBZDC operon, the upstream mmoR and mmoG genes are also indicated. These encode, respectively, a putative {sigma}54-dependent transcriptional regulator and molecular chaperon that (while not part of the mature sMMO complex) are needed for expression of active sMMO in vivo (39). PCR primers for amplification of 'mmoC were mmodel-1 (5'-ATA ATA GGA TCC ATC GTC ATC GAG ACC GAG GAC G-3'; BamHI site underlined) and the M13 universal sequencing primer (3'-GTA AAA CGA CGG CCA GT-5'). Deletion of mmoXYBZD and the upstream region of the mmoC gene was effected by introducing pMD-Mdel3 into M. trichosporium by conjugation (25) and selecting exconjugants by using gentamicin. M. trichosporium SMDM, the exconjugant in which the sMMO-encoding operon had been deleted via double homologous recombination, was selected on the basis of its sMMO-negative phenotype as determined by the naphthalene oxidation test (1, 2), together with its Gmr KnS phenotype. The image is not drawn to scale.

 
Site-directed mutagenesis and preparation of cells expressing the mutant enzymes.
Site-directed mutagenesis of the mmoX gene, encoding the {alpha} subunit of the sMMO hydroxylase, was performed via the four-primer overlap extension method (15) as described previously (38). Primers used for mutagenesis are given in Table 2. Dye termination sequencing was used to confirm the absence of unwanted mutations from PCR-derived portions of the cloned mutant sMMO genes. The mutant mmoX genes, assembled into an intact mmoXYBZDC operon in the sMMO expression plasmid pTJS175 (38), were introduced into M. trichosporium SMDM by conjugation (25, 38). The recombinant sMMO strains were initially grown on NMS plates containing antibiotics (gentamicin and kanamycin) and screened for sMMO activity by using the naphthalene oxidation test (1, 2). Cells for more-detailed analysis were prepared by growing the recombinant M. trichosporium cells in a fermentor to an OD600 greater than 5 in order to induce mutant enzyme expression from the native sMMO promoter in pTJS175, which is induced by low copper-to-biomass ratio in the culture.


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  		TABLE 2. Primers used for site-directed mutagenesis

 
sMMO assays.
sMMO is the only enzyme system expressed in M. trichosporium that is able to oxygenate aromatic hydrocarbon substrates. As a result, assays with these substrates can be performed using whole-sMMO-expressing cells. The semiquantitative naphthalene oxidation test, which is based on the derivatization of the naphthol products of naphthalene oxidation to pink/purple diazo dyes, was performed on cultures in liquid medium and on agar plates as described previously (1, 2). After oxidation of aromatic hydrocarbons by cells expressing mutant or wild-type sMMO, product analysis was performed as follows. Methanotroph cells were resuspended in 25 mM MOPS (morpholinepropanesulfonic acid) buffer (pH 7.0) to an OD600 of greater than 5. Each assay was conducted with 5 ml of cell suspension or 10 ml where biphenyl was the substrate. Oxidation substrate was added, together with sodium formate (10 mM), to ensure a plentiful supply of the reducing equivalents required by sMMO. The amounts of oxidation substrate used were 50 µl for liquid substrates (toluene or ethylbenzene) and 1 mg for solid substrates (naphthalene, biphenyl, phenanthrene, or anthracene). The reaction mixture was incubated aerobically at 30°C for 48 h with shaking (250 rpm) in a closed 30-ml glass vial held in a horizontal position. The hydroxylated products were extracted into 1.0 ml of diethyl ether, which was then evaporated to a volume of 50 µl before analysis. The products of toluene and biphenyl oxidation were characterized by means of gas chromatography (GC), using a 6890 GC apparatus (Hewlett-Packard) fitted with a Stabilwax capillary column with a Carbowax polyethylene glycol coating (50 m by 0.32 mm; coating thickness, 1 µm) and coupled to a flame ionization detector. The column temperature at the beginning of the separation was held at 100°C for 5 min, after which it was ramped to 180°C at 2°C min–1 and held for 15 min at 180°C. The flow rate of carrier gas (nitrogen) was 1.5 ml min–1, and the split ratio was 10:1. When the substrate was naphthalene or ethylbenzene, products were analyzed via GC-mass spectrometry (MS) using a 5890 GC (Hewlett-Packard) coupled to a Trio-1 MS. Here, the GC was fitted with a Hewlett-Packard HP-5 column with a (5% phenyl) methyl polysiloxane coating (50 m by 0.32 mm; coating thickness, 0.25 µm) and operated with a carrier gas (nitrogen) flow rate of 1.5 ml min–1. With ethyl benzene as the substrate, the split ratio was 30:1 and the column temperature was ramped from 80°C to 250°C at 4°C min–1 and held for 1 min at 250°C. When naphthalene was the substrate, the separations were carried out without a split and the column temperature was ramped from 80°C to 126°C at 10°C min–1, from 126°C to 129°C at 0.1°C min–1, and then from 129°C to 250°C at 10°C min–1. When biphenyl was the substrate, the products were separated via a PerkinElmer autosystem GC using the Hewlett-Packard HP-5 column (details described above) coupled to a flame ionization detector. The separations were carried out without a split. The carrier gas (nitrogen) flow rate was 1 ml min–1, and the column temperature was ramped from 90°C to 190°C at 5°C min–1, held at 190°C for 1 min, and then ramped to 280°C at 10°C min–1. Products were identified by comparison of retention times and mass spectra to authentic standards.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of a new sMMO-deleted methanotroph expression host for recombinant sMMO enzymes.
Previous mutants of the hydroxylase component of sMMO were expressed in the sMMO-negative mutant of M. trichosporium known as mutant F (38). The chromosomal copy of the sMMO operon in mutant F (26) has a deletion of a 1.2-kb fragment that encompasses the portion of mmoX coding for amino acids Val 112-Thr 508. This limits the region of the sMMO operon that can be mutated to the portion of mmoX (encoding the {alpha} subunit of the hydroxylase) that is deleted from mutant F, because mutations outside this region could be repaired by homologous recombination between the plasmid carrying the mutant sMMO operon and the remainder of the chromosomal copy. The mutagenesis target during this study, the portion encoding Leu 110, lies just outside this region, so it was in principle possible that mutations could be repaired by homologous recombination with the chromosome. Furthermore, for future studies we wished to produce an expression host for recombinant sMMO that would allow mutagenesis anywhere within the hydroxylase component (encoded by mmoX, -Y, and -Z). The new expression host, M. trichosporium SMDM (for soluble-methane-monooxygenase-deleted mutant), in which the first five genes of the sMMO operon (mmoXYBZD, including all the structural genes for the hydroxylase and protein B) had been fully deleted by marker exchange mutagenesis, was therefore constructed (Fig. 1). The deletion of the intended portion of the sMMO-encoding operon and double recombination into the chromosome were confirmed by restriction digestion of chromosomal DNA and digestion with ClaI and SphI, followed by Southern blotting and probing with probes specific to mmoR, mmoX, and mmoY (data not shown). Strain SMDM exhibited the expected sMMO-negative phenotype, as judged by a negative naphthalene oxidation test result under low-copper conditions, which induced sMMO expression and under which a parallel control with wild-type M. trichosporium OB3b showed strong naphthalene oxidation activity.

The suitability of strain SMDM as a host for expression of recombinant sMMOs was established by introducing pTJS175, containing the wild-type sMMO operon, into SMDM to give strain SMDM-sMMO (Table 1). Growth on NMS agar medium containing a low level of copper(II) (0.4 µM) to induce sMMO yielded colonies that gave strong positive results in the naphthalene oxidation test, confirming the expression of the recombinant sMMO.

Mutagenesis of the leucine gate residue produced mutants that gave unusual naphthalene oxidation test results.
Four mutants of the proposed gating residue Leu 110 that gave a range of amino acid bulk and functionality results at this position were constructed. Mutations to Gly (less bulky; more flexible), Cys (less bulky), Arg (slightly more bulky; positively charged), and Tyr (more bulky; hydroxylated) were constructed, confirmed by DNA sequencing, and expressed in the M. trichosporium SMDM homologous expression system, as described in Materials and Methods. When the four mutant strains were grown in liquid culture at a low copper-to-biomass ratio, which induces expression of sMMO, all four gave positive naphthalene test results, confirming that all the mutant clones produced mutant enzymes that were active with naphthalene as the substrate. The L110G and L110C mutants gave positive naphthalene test results where the purple diazo dye product was, like that from the wild type, stable for more than 10 min. The colors produced by the L110R and L110Y mutants, however, were stable for less than 1 min and appeared pink compared to the purple seen with the wild type (data not shown). Although we had previously observed mutants of sMMO where the naphthalene test gave a very weak color change, indicating a low-activity or unstable enzyme (38), this was the first time that we had observed a mutant where the stability (and color) of the naphthalene test result was markedly changed.

The L110R and L110Y mutants of sMMO show inverted regioselectivity with naphthalene compared to the wild type.
It was reasoned that the qualitative difference in the stabilities of the naphthalene test colors of the mutant and wild-type enzymes was most likely due to an alteration in the position(s) of oxygenation in the products of naphthalene oxidation by the L110R and L110Y mutants. GC-MS analysis (Table 3) confirmed that there were significant alterations in the distribution of monohydroxylated products from the mutant enzymes. Whereas the wild-type enzyme and the L110C mutant gave a slight excess of 2-naphthol over 1-naphthol, this regioselectivity was reversed in the other three mutants, and the two mutants that gave the unstable naphthalene test color (L110R and L110Y) yielded the smallest relative amounts of 2-naphthol. Other possible products, such as dihydroxylated moieties, were not detected. A separate experiment using 10 µM solutions of the two isomers of naphthol and the same concentration of tetrazotised o-dianisidine as used in the naphthalene test for sMMO activity revealed that the diazo dye produced by 1-naphthol forms more quickly and decays more quickly than that produced by 2-naphthol (Fig. 2). Thus, the difference in the regioselectivity of the mutants accounts for the visual differences in the results of their naphthalene tests.


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  		TABLE 3. Products of naphthalene oxidation by the mutants

 

Figure 2
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  		FIG. 2. Stabilities of the colored diazo compounds formed by reaction of tetrazotised o-dianisidine with 1-naphthol (solid line) or 2-naphthol (dotted line).

 
The L110 mutants showed a relaxed regioselectivity and generated novel products with substituted monoaromatic substrates and biphenyl.
With the substituted monoaromatic substrates toluene and ethylbenzene, the wild-type sMMO exhibits a mixture of side chain hydroxylation and ring hydroxylation at the p position, with ring hydroxylation predominating with toluene and side chain hydroxylation with ethyl benzene. With toluene as the substrate, all the mutations at position 110 showed relaxed regioselectivity (Table 4), with the appearance of significant amounts of m-cresol, which is not seen with the wild type. Interestingly, when Leu 110 was replaced by the smaller glycyl and cysteinyl residues, with toluene as the substrate, more benzyl alcohol was produced than p-cresol. When ethylbenzene was the substrate, all the mutants showed very large shifts from side chain to ring hydroxylation and at least one new product (2-ethylphenol) was observed with each mutant (Table 5). 3- and 4-ethylphenol were not resolved in these experiments, so it is not clear whether the product 3-ethylphenol was produced. With biphenyl as the substrate, the wild type produced predominantly 4-hydroxybiphenyl. Mutants with the larger tyrosine or arginine residue at position 110 produced substantially more 2-hydroxybiphenyl than the wild type. The mutants with the smaller residues (glycine and cysteine) at this position yielded the new product 3-hydroxybiphenyl (Table 6).


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  		TABLE 4. Products of toluene oxidation by the mutants

 

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  		TABLE 5. Products of ethyl benzene oxidation by the mutants

 

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  		TABLE 6. Products of biphenyl oxidation by the mutants

 
Neither the mutants nor the wild type oxidized triaromatic hydrocarbons.
Cells expressing wild-type sMMO and all four mutations at position 110 were initially screened for oxidation of the triaromatic compounds phenanthrene and anthracene in the same manner used for the semiquantitative naphthalene assay, by addition of tetrazotised o-dianisidine, which gave colored diazo dyes after reaction with the hydroxylated products. No visible color changes were seen from the mutants or the wild type, although a 9-phenanthrol control gave a colored product with a detection threshold of 5 µM. GC-MS was used to analyze the assay reaction mixtures that had been incubated for 30°C for 48 h, as described in Materials and Methods, but no hydroxylated products could be found from either of the triaromatic substrates with wild-type M. trichosporium OB3b or any of the mutant strains. Presuming that phenanthrene and anthracene are able to diffuse into the cells, these results strongly suggest that neither wild-type sMMO nor the mutants are able to oxidize these bulky aromatic substrates.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Further development of the expression and mutagenesis system.
Mutagenesis of the hydroxylase component of sMMO requires the use of an especially developed expression system because attempts to date to obtain expression of active hydroxylase in E. coli have been unsuccessful (44). Expression of active sMMO has been reported to occur in various heterologous systems (16, 17, 24) as well as the homologous system used here. The previous work that we performed using the homologous expression system for sMMO, which enabled the first mutagenesis of the active site of the enzyme (38), utilized a homologous expression host (M. trichosporium mutant F [26]) from which only part of the mmoX gene was deleted, thus limiting the region of the hydroxylase that could be mutagenized. The new strain developed here, M. trichosporium SMDM, like mutant F, is a homologous expression host that gives expression of recombinant wild-type sMMO comparable to that from the parental strain M. trichosporium OB3b. Moreover, strain SMDM not only allows mutagenesis anywhere within the active site-containing {alpha} subunit, including Leu 110, but also allows mutagenesis of all components of the sMMO-encoding operon except the mmoC reductase, i.e., the hydroxylase (mmoXYZ), protein B (mmoB), and mmoD genes, whose product may have a role in sMMO complex assembly (27).

Regioselectivity of the wild-type sMMO with aromatic substrates.
Our results with M. trichosporium expressing recombinant wild-type sMMO confirmed previous results concerning the regioselectivity of sMMO with aromatic substrates. If the substrates are considered to be roughly rectangular, the wild-type enzyme shows a strong preference for hydroxylation nearest to the two short sides of the rectangle. Thus, naphthalene gives primarily 2-naphthol, biphenyl gives predominantly 4-hydroxybiphenyl, and toluene and ethyl benzene undergo a mixture of side chain and 4-position hydroxylation on the benzene ring (2, 3, 14).

Mutations at Leu 110 in sMMO relax regioselectivity but do not extend activity to triaromatic hydrocarbons.
The mutant strains showed lower rates of total product accumulation than the wild type, ranging from 2.5 to 63% of the wild-type activities (Tables 4 and 5). Examining the data from ethyl benzene and toluene as substrates, we did not find a clear correlation between the property of the amino acid and the reduction in activity, and these results may also reflect differences in expression and stability between the mutants. Analysis of the proportions of products formed from the aromatic substrates, however, yielded information that is not biased by the level of expression.

As detailed in the introduction, previous work on homologous monooxygenases that naturally oxidize aromatic substrates has variously indicated effects of mutations at the position equivalent to Leu 110 of the hydroxylase {alpha} subunit of sMMO on regioselectivity and substrate range. In the case of sMMO, none of the mutants, including those with substantially smaller residues at position 110, had measurable activity toward the triaromatic substrates phenanthrene and anthracene. However, all had substantial changes in regioselectivity, showing a shift away from "terminal" oxidation of aromatic substrates, with increasing proportions of 1-naphthol from naphthalene and 2- and 3-hydroxy products from substituted monoaromatics and biphenyl. This result was to some extent unexpected, since a "gating role" for Leu 110 suggested that mutations to smaller residues would be likely to let larger substrates into the active site. There was some correlation between the changes in product distribution and the size of the residue at position 110. The shift in regioselectivity of naphthalene oxidation was greatest in the mutants with the largest residues (Tyr and Arg) at position 110. Conversely, the novel product from biphenyl oxidation, 3-hydroxybiphenyl, is seen only in the mutants with the smaller residues (Gly and Cys) at this position. The shift from side chain to ring hydroxylation of toluene was greatest in the mutants with smaller residues (Gly and Cys) at position 110, and the corresponding shift in regioselectivity in oxygenation of ethyl benzene was almost 2 orders of magnitude greater than that seen with any of the other mutants. In the homologous soluble butane monooxygenase, the mutation G113N, which is near the "leucine gate" (Leu 109 in butane monooxygenase), also led to a shift in regioselectivity away from the ends of the substrate molecules, in this case giving rise in the regiospecificities of oxygenation of butane and propane from predominantly terminal to predominantly subterminal (13).

In monooxygenases of the naphthalene dioxygenase family, a residue at the narrowest part of the active-site pocket (Phe 352 in naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4) (18) has also been shown, via mutagenesis studies (19, 29), to influence substrate range and regioselectivity. X-ray crystallography of an enzyme-product complex indicated that the product (and presumably also the substrate) contacts Phe 352 when bound in the active site (6). Unfortunately, there are currently to our knowledge no structural data for sMMO or its homologues complexed with aromatic substrates, but the situation with Leu 110 in these enzymes seems likely to be different, since this residue is on the edge of the active site and less likely to contact the substrate at the moment of oxygenation.

Our data indicate that Leu 110 is an important residue in determining the precision of regioselectivity of sMMO with aromatic substrates. The various mutations that we have made at this position relax the regioselectivity for aromatic substrates, which could be advantageous in activating polyaromatic pollutants for biological breakdown by consortia of microorganisms. While our results do not allow us to judge whether Leu 110 functions as a substrate gate, they strongly suggest that Leu 110 is not the limiting factor for the size of an aromatic substrate that can enter the active site. It may be that the important role that Leu 110 has in determining the catalytic specificity of the enzyme may be mediated via conformational changes of the type already shown to affect this residue in various crystal forms of the enzyme (30). In the future, we propose to undertake detailed kinetic and structural analysis of the mutants to determine how their effect on the regioselectivity of the enzyme is mediated.


   ACKNOWLEDGMENTS
 
We are grateful to Joan Hague and Keith Richards (Sheffield Hallam University) for advice and technical assistance with GC and GC-MS. We thank Julie Scanlan (Warwick) for technical support.

This work was supported by the Biotechnology and Biological Sciences Research Council through a research grant (reference no. BB/C003276/1 and BB/C00339X/1) to J.C.M. and T.J.S.


   FOOTNOTES
 
* Corresponding author. Mailing address: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom. Phone: 44 114 225 3042. Fax: 44 114 225 3066. E-mail: t.j.smith{at}shu.ac.uk Back

{triangledown} Published ahead of print on 17 August 2007. Back

{dagger} E. Borodina and T. Nichol made equal contributions to this work. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Applied and Environmental Microbiology, October 2007, p. 6460-6467, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00823-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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