On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

DmpK from Pseudomonas sp. strain CF600 represents a group of proteins required by phenol-degrading bacteria that utilize a multicomponent iron-containing phenol hydroxylase. DmpK has been overexpressed in Escherichia coli and purified to homogeneity; it lacks redox cofactors and was found to strongly inhibit phenol hydroxylase in vitro. Chemical cross-linking experiments established that DmpK binds to the two largest subunits of the oxygenase component of the hydroxylase; this may interfere with binding of the hydroxylase activator protein, DmpM, causing inhibition. Since expression of DmpK normally appears to be much lower than that of the components of the oxygenase, inhibition may not occur in vivo. Hence, the interaction between DmpK and the oxygenase manifested in the inhibition and cross-linking results prompted construction of E. coli strains in which the oxygenase component was expressed in the presence and absence of a low molar ratio of DmpK. Active oxygenase was detected only when expressed in the presence of DmpK. Furthermore, inactive oxygenase could be activated in vitro by adding ferrous iron, in a process that was dependent on the presence of DmpK. These results indicate that DmpK plays a role in assembly of the active form of the oxygenase component of phenol hydroxylase.

There is currently much interest in the biochemistry of oxygenases, which control the entry of toxic compounds into catabolic pathways that allow microorganisms to break them down to carbon dioxide and water. A recently discovered class of aromatic oxygenases includes phenol (1)(2)(3) and toluene (4 -6), monooxygenases that are in many ways similar to the relatively well characterized binuclear iron center-containing enzyme, methane mononoxygenase (reviewed in Ref. 7). Various types of experimental evidence indicate that these enzyme systems all utilize an FAD/[2Fe-2S] center containing reductase, a low molecular weight activator protein (similar to MmoB), and a heteromultimeric (␣␤␥) oxygenase component.
The degradation of phenol by Pseudomonas sp. strain CF600 requires the participation of polypeptides encoded by the 15 genes of the dmp operon (3), and is initiated by the hydroxyla-tion of phenol to catechol. The gene products of dmpLMNOP were found to be necessary for phenol hydroxylase activity in an in vitro assay containing phenol, NADH, and Fe 2ϩ (2). The reductase component of this enzyme is encoded by dmpP, the oxygenase component by dmpLNO, and the activator protein by dmpM (2,3). 1 Polypeptide requirements for enzyme activity are summarized in Scheme 1.
An additional gene, dmpK, is located immediately upstream of the phenol hydroxylase genes in the operon. The presence of dmpK was found to be necessary to allow growth on phenol of a strain of Pseudomonas harboring dmpLMNOP together with enzymes necessary to catabolize catechol, the product of phenol hydroxylase (1). However, the function of the 10.5-kDa gene product remained obscure.
Nucleotide sequencing has since revealed the presence of genes encoding similar proteins clustered together with phenol hydroxylase genes in other strains of Pseudomonas (96% identity) (8,9) and in Acinetobacter (52% identity over 68 amino acids) (10). Southern hybridization experiments also established the presence of DNA homologous to dmpK in a number of phenol-degrading marine bacteria, and in the archetypal phenol degrader, Pseudomonas U (11). Furthermore, a gene (tbmA) associated with a multicomponent toluene monooxygenase system exhibits 38% identity, over 55 amino acids, with DmpK (12). The functions of these DmpK homologues have not been defined, nor have any homologous proteins of known function been identified in sequence data base searches.
In this report we have overexpressed and purified DmpK, and characterized its interaction with the oxygenase component of phenol hydroxylase. The data are consistent with the involvement of DmpK in iron-dependent assembly of an active oxygenase.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were reagent grade or higher. The purification from phenol-grown Pseudomonas sp. strain CF600 of DmpM and the oxygenase component (DmpLNO) of phenol hydroxylase will be described elsewhere. 1 The reductase component, DmpP, was purified using a previously published method (2). Catechol 2,3-dioxygenase was purified essentially as described previously (13) from Escherichia coli harboring pMMB26 (14).
Plasmid Constructs and Strains-Strains and plasmids used in this study are listed in Table I. DNA manipulations were performed according to standard techniques (20). In order to express DmpK alone and as a fusion protein with TrpE, the dmpK gene was first amplified by polymerase chain reaction as an NdeI to BamHI fragment (base pairs 745-1052; Ref. 1) and cloned into a pBluescript (Stratagene) derivative, in which an NdeI site had previously been introduced into the polylinker, to generate pVI202. Both strands of the resulting fragment were sequenced to ensure that no mutations had been introduced.
The dmpK gene was introduced into a pET expression vector (16), and the resulting plasmid was used for overexpression of DmpK. The NdeI-BamHI fragment of pVI202 was cloned between these sites of pET3a to generate pVI203 with dmpK under control of the T7 promoter of the vector.
A linker was introduced into the NdeI site of pVI202, generating pVI204, to facilitate cloning of dmpK as an EcoRI-BamHI fragment in frame with the trpE gene in pATH11 (19). The resulting plasmid, pVI205, encodes the first 330 amino acids of TrpE fused to the 92 residues of DmpK with 3 residues (Glu, Phe, and Arg) inserted at the junction.
In order to compare the activities of the oxygenase component (Dm-pLNO) of phenol hydroxylase expressed in the presence and absence of the dmpK gene product, dmpK was inserted into pVI293 in place of dmpP to generate pPOW100; this plasmid expresses dmpLNOK from the inducible tac promoter of the pMMB66 vector. A BglII-EcoRI fragment from pVI293 (encompassing base pairs 3728 -5448 of the phenol hydroxylase coding region; Ref. 1) was excised and cloned between these sites in pET3a. An NdeI site was introduced into this plasmid at the ATG start codon of dmpP using the U.S.E. mutagenesis kit (Pharmacia). The NdeI-EcoRI fragment of the resulting plasmid, encompassing dmpP, was then replaced by the NdeI-EcoRI fragment, encoding dmpK, from pVI203. Finally, the BglII-EcoRI fragment was re-excised and introduced between these sites of pVI293 to generate pPOW100.
A related plasmid, pPOW101, was constructed in parallel, except that the NdeI-EcoRI fragment introduced from pVI203 was substituted by the NdeI-EcoRI fragment from the vector, pET3a. The BglII-EcoRI fragment of the resulting plasmid was then cloned between these sites in pV1293 to generate pPOW101. This plasmid thus expresses only dmpLNO, together with an unrelated 16-amino acid peptide, from the inducible tac promoter of the pMMB66 vector.
Expression Conditions-Recombinant DmpK was expressed after introducing pVI203 into E. coli BL21 (DE3). Fresh transformants were used to inoculate Luria broth (LB) containing ampicillin (100 g/ml) and glucose (0.4%), and the cells were then grown at 37°C with vigorous shaking to OD 650 ϭ 0.6 -0.8. Cells were then harvested by centrifugation, washed with prewarmed medium, and resuspended in an equal volume of LB containing ampicillin (100 g/ml); in some cases, this step was omitted with little noticeable change in DmpK expression levels. After incubation for an additional 1 h, cultures were supplemented with IPTG 2 (0.5 mM) to induce expression of the host-encoded T7 polymerase that is under the control of the P tac promoter. After an additional 3 h of growth, cells were harvested, washed, and stored as a paste at Ϫ80°C until use.
High levels of expression were also achieved in cells that were grown using forced aeration in a fermentor. In this case, an overnight culture (400 ml) of cells grown in LB containing carbenecillin (100 g/ml) was inoculated into a fermentor filled with 16 liters of LB broth containing ampicillin (100 g/ml). Induction with IPTG (at OD 650 ϭ 0.85) and harvest were as described above.
The procedure for expression of TrpE-DmpK was essentially as published previously (19). In brief, M9 minimal medium (20) containing ampicillin (100 g/ml) and casamino acids (0.5%) was inoculated with fresh transformants, and the culture grown at 37°C with vigorous shaking for 3 h. Cells were then harvested, washed with the same medium lacking tryptophan, and resuspended in an equal volume of fresh medium: after growth for 1 h, cultures were supplemented with indoleacetic acid (20 g/ml) to induce expression from the P trp promoter. After incubation of the cultures for an additional 3 h, they were harvested by centrifugation, washed with M9 medium, and then stored as a paste at Ϫ80°C until further use.
In studies of the oxygenase component expressed from pPOW100 and pPOW101, overnight cultures were grown in LB containing ampicillin or carbenecillin (100 g/ml) to OD 650 ϭ 0.7-1.0, and then supplemented with IPTG (0.5 mM). After incubation for an additional period, cells were harvested by centrifugation, washed once with 20 mM MOPS, pH 7.4, and then stored as a paste at Ϫ80°C until further use.
Immunological Methods-To obtain high titer antisera to the low molecular weight DmpK polypeptide (92 amino acids), a strategy employing both the TrpE-DmpK fusion and DmpK proteins was employed. Crude extracts of BL21(DE3)pVI203 (expressing DmpK) and BL21(DE3)pVI1205 (expressing TrpE-DmpK, 425 amino acids) were prepared by sonication of cells resuspended in 50 mM Tris-HCl, pH 7.5. After removal of cell debris by ultracentrifugation, soluble proteins were separated using 10 -20% SDS-PAGE, and the polypeptide bands corresponding to the proteins of interest were excised and homogenized in phosphate-buffered saline. These preparations were used for rabbit antisera production by sequential immunization with TrpE-DmpK (2 injections) and DmpK (3 injections), using methods described previously (21).
For Western blot analysis, proteins were transferred from 10 -20% SDS-PAGE gradient gels to polyvinylidene difluoride membranes using standard electroblotting methods (22). The blots were developed using an immunoblot assay kit (Bio-Rad) with goat anti-rabbit IgG conjugated to alkaline phosphatase. The serum containing primary antibody was diluted 1:1000 for use.
Purification of DmpK-The buffer used during the purification procedure was 50 mM Tris-HCl, pH 8.0, containing 10% glycerol and 1 mM dithiothreitol (''Buffer A''). All purification procedures were performed at 4°C. The presence of DmpK in column fractions was monitored using SDS-PAGE.
Crude Extract Preparation-Crude extract of BL21(DE3)pVI203 (expressing DmpK) was prepared by sonication of a suspension of cells (51 g, wet weight) in buffer A (100 ml). A few milligrams of DNase I were then added, and the cell suspension was incubated at 4°C for 15 min with occasional stirring. The cell suspension was then sonicated in 15-s bursts (five times), followed by centrifugation at 70,500 ϫ g for 1 h. The supernatant was decanted carefully and used for further purification.
Ion Exchange Chromatography-The crude extract was loaded (6 ml/min) onto a Fast-Flow DEAE-Sepharose column (36 ϫ 2.6 cm) equilibrated with Buffer A. The column was then washed with approximately 200 ml of this buffer, followed by a linear gradient of 0 -0.2 M NaCl in Buffer A (1500 ml). Fractions (12 ml) containing DmpK were combined and brought to 80% saturation with ammonium sulfate, and, after 15 min on ice, the precipitate was collected by centrifugation. Precipitated protein was dissolved in Buffer A (50 ml) containing ammonium sulfate (1 M) in preparation for the next step.
Hydrophobic Interaction Chromatography-One-third of the sample from the previous step was loaded (2 ml/min) onto a Phenyl-Sepharose (Pharmacia) High Performance column (36 ϫ 2.6 cm) equilibrated with buffer A containing ammonium sulfate (1 M). The column was washed with 50 ml of this buffer and then developed with a linear gradient of ammonium sulfate (1-0 M in 1200 ml). Two peaks containing DmpK eluted, and fractions from each peak were pooled and subjected separately to the next, and final, purification step.
Gel Filtration Chromatography-In preparation for this step, proteins from the previous step were concentrated by precipitation with ammonium sulfate, as detailed above. Precipitated protein was redissolved in Buffer A and loaded (1 ml/min) onto a Sephacryl S-300HR column (95 ϫ 2.5 cm) equilibrated with Buffer A. Proteins were eluted using the same buffer, and fractions (12 ml) were collected. Those fractions found to contain DmpK were combined, concentrated by ultrafiltration using an Amicon PM-10 membrane, and then stored as small aliquots at Ϫ80°C.
On occasion, DmpK collected from the DEAE-Sepharose column was concentrated by precipitation with ammonium sulfate and applied directly to the gel filtration column. In these cases, a major peak eluted from the gel filtration column early on, and a minor one eluted much later; both contained DmpK, as judged by SDS-PAGE. However, preparations were less pure than those obtained when the Phenyl-Sepharose step was included.
Chemical Cross-linking Experiments-Cross-linking reactions were carried out at room temperature in 50 mM MOPS buffer, pH 7. Control reactions were also run in which cross-linker or proteins were omitted. Cross-linkers were added to protein solutions at room temperature, and the mixtures were incubated for 30 -60 min, after which reactions were quenched by the addition of SDS-PAGE sample buffer. Cross-linked samples were then analyzed by SDS-PAGE and/or Western blotting. Although both prestained (Bio-Rad) and unstained (Pharmacia) molecular weight markers were used, the reported molecular weights were estimated by comparison of R F values with those of the non-prestained standards.
Analytical Methods-Protein concentrations were generally estimated using the bicinchoninic acid (BCA) method (Pierce) following the 60°C protocol supplied by the manufacturer and modified when necessary to remove interfering substances (23). Bovine serum albumin was used as the standard.
Electrospray mass spectrometry was done using a Finnigan SSQ 7000 single quadrupole mass spectrometer. Samples were prepared by exchanging the buffer for distilled water using Bio-Spin columns (Bio-Rad). Samples were further diluted into methanol/water/acetic acid (50%/50%/0.5%) before infusion into the spectrometer.
Amino-terminal sequencing of purified DmpK was done by Dr. Per-Ingvar Ohlsson, Department of Medical Chemistry, Umeå University, using an Applied Biosystems model 477A peptide sequencer.
Circular dichroism spectra were collected at room temperature using a Jasco J-710 CD spectrometer. UV-visible measurements were performed using a Philips PU8710 spectrophotometer.
Iron concentrations were estimated colorimetrically using o-phenanthroline after precipitation of protein with trichloroacetic acid (26).
Where indicated, ferrous ammonium sulfate was added to assays, from a 10 mM stock solution in 50 mM HCl, to a final concentration of 5 M. The addition was made immediately after the addition of the oxygenase preparation, and assay mixtures were then incubated for 2 min before the addition of phenol to initiate the reaction. Addition of DmpK was made after the addition of oxygenase preparation, and prior to the addition of ferrous ammonium sulfate.

RESULTS
Expression of DmpK by Pseudomonas sp. Strain CF600 -Although the phenol hydroxylase polypeptides, DmpLMNO, are expressed at high enough levels in Pseudomonas sp. strain CF600 to be readily distinguished in crude extracts run on SDS-polyacrylamide gels (in comparison with extracts from uninduced cells), it was impossible to detect DmpK on SDS-PAGE gels by inspection. However, Western blotting revealed the presence of DmpK in crude extracts from phenol-grown cells but not in extracts from acetate-grown cells (data not shown). The low level of expression of DmpK is consistent with the suboptimal position of its poor ribosome binding site (1) and has physiological implications, discussed below.
In order to express sufficient DmpK to allow purification, dmpK was cloned and expressed in E. coli from the strong viral T7 promoter in pVI203, as described under ''Experimental Procedures.'' Purification and Properties of DmpK-Recombinant DmpK was purified from E. coli harboring pVI203 using Fast-Flow DEAE chromatography, followed by chromatography on gel filtration and Phenyl-Sepharose columns. Samples from each stage of a representative purification are shown in Fig. 1. Approximately 125 mg were obtained from the equivalent of 17 g (wet weight) of cell paste.
The protein exhibited some heterogeneity on the gel filtration and Phenyl-Sepharose columns. When the Phenyl-Sepharose step was omitted and the eluate from the DEAE column was applied directly to the gel filtration column, high and low molecular weight forms of DmpK were observed. The high molecular weight form predominated, with more than 90% of the total DmpK eluted from the column existing in this form. Two broad peaks of similar size were observed when the eluate from the DEAE column was instead chromatographed on the Phenyl-Sepharose column; one eluted early in the salt gradient, and the other eluted immediately after. Protein from each peak was further purified by gel filtration chromatography; the two fractions behaved identically on this column, each eluting at the position of the high molecular weight form referred to above (data not shown).
All fractions of DmpK were essentially pure, as judged by SDS-PAGE (e.g. Fig. 1) and electrospray mass spectrometry (not shown), and were indistinguishable using either of these techniques or native gel electrophoresis; circular dichroism spectroscopy of the different fractions showed only minor differences. The molecular weight obtained by electrospray ionization-mass spectroscopy was 10,451 Ϯ 1 mass unit, which corresponds well with the molecular weight deduced from the nucleotide sequence (10,586), assuming that the amino-terminal methionine had been removed. This was confirmed by amino-terminal sequencing of the purified protein. The native mo- lecular weight of the protein was estimated to be approximately 37,000 using a calibrated high pressure liquid chromatography gel filtration column: thus, DmpK appears to exist mainly as a trimer or a tetramer. Purified DmpK fractions were colorless, with a peak observed only in the UV region of the spectrum (data not shown). Therefore, the purified protein does not contain redox-active prosthetic groups such as flavin, heme, or iron-sulfur centers. Additionally, preparations of DmpK were found to contain only traces of iron, so the purified protein does not contain a spectroscopically invisible iron center either.
Inhibition of Phenol Hydroxylase Activity by DmpK-The dose-dependent effects of adding a purified preparation of DmpK to phenol hydroxylase assays, in the presence and absence of DmpM, are summarized in (Fig. 2). DmpK-dependent inhibition of the hydroxylase in the presence of DmpM was dramatic, with inhibition occurring at low levels of DmpK. The phenol hydroxylase-catalyzed reaction was much slower in the absence of DmpM, but was still readily detectable, and appeared to be insensitive to the presence of DmpK (Fig. 2). These results suggest that DmpK can interfere with the interaction between the oxygenase component (DmpLNO) and DmpM, the activator protein. However, since the in vivo ratio of DmpK: DmpLNO appears to be very low (see above), inhibition of the hydroxylase is of questionable physiological significance.
Direct Evidence for Interaction between DmpK and Phenol Hydroxylase-Since the inhibition experiments suggested that DmpK interacts with one or more of the phenol hydroxylase polypeptides (excluding DmpP), chemical cross-linking was used to probe potential interactions. Direct evidence for the interaction of DmpK with the two larger subunits of the hydroxylase was obtained using the homobifunctional N-hydroxysuccinimide-ester cross-linker, BS 3 . DmpK (10.5 kDa) appeared to be cross-linked to itself by this reagent, as indicated by three prominent bands of cross-linked products with apparent molecular masses ranging from 19.5 to 24 kDa observed upon Coomassie Blue staining and Western blotting using a DmpK-specific antibody (Fig. 3). These bands are likely to represent DmpK dimers; the range of products observed may be ascribed to additional modifications and/or internal cross-linking of the dimeric protein, either of which could alter the mobility of cross-linked products. An additional band visible on the Western blot (Fig. 3b, lane D) just below the 35.1-kDa marker is consistent with the gel filtration results, which suggested that the protein is tri-or tetrameric. However, the yield of this cross-linked product is low, and it is not visible on the Coomassie-stained gel.
Upon exposure to BS 3 , the oxygenase component of phenol hydroxylase, which is composed of subunits running at 54, 36, and 13.5 kDa on SDS-PAGE (molecular masses predicted from the gene sequences are 60.5 (DmpN), 38.5 (DmpL), and 13.2 kDa (DmpO); see Ref. 1), gave rise to products running at 92 and 120 kDa (Fig. 3a). The 92-kDa band is likely to be the cross-linked product of DmpL (36 kDa, observed) and Dmp N (54 kDa, observed). It is difficult to identify the 120-kDa product without additional information.
In the presence of BS 3 a mixture of DmpK and oxygenase component gave rise to new cross-linked products (Fig. 3a), each of which reacted with the DmpK-specific antibody (Fig.  3b). The major products had molecular masses of 46 kDa and 69 kDa, which most likely represent DmpK linked to DmpL (12.5 plus 36 kDa, observed) and DmpK and DmpN (12.5 plus 54 kDa, observed), respectively. Two larger complexes containing DmpK were also observed (Fig. 3b); that running at 96 kDa most likely represents a complex of DmpKLN. It should be noted that although DmpK can clearly bind to polypeptides of the oxygenase complex, it does not co-purify with it (Fig. 3b,

lane E).
These data provided clear evidence that DmpK binding sites exist on the two largest subunits of the hydroxylase. In light of the cross-linking and inhibition results, the effects of co-expressing it at a low level (i.e. at far less than a molar ratio) with the oxygenase (DmpLNO) were examined in order to provide more information about a potential physiological role.
Expression of the Oxygenase Component of Phenol Hydroxylase in the Presence and Absence of DmpK-In a previous study, recombinant phenol hydroxylase was expressed in the presence and absence of DmpK, and phenol hydroxylase activity was measured in the presence of Fe 2ϩ (2). Since then, we have developed a sensitive assay for phenol hydroxylase activity in the absence of added iron and here re-examine the activity of the recombinant oxygenase component when expressed with or without DmpK.
Preliminary experiments (data not shown) indicated that expression levels of the oxygenase component polypeptides, DmpLNO, differed substantially in two earlier plasmid constructs, pVI290 and pVI293, which express DmpKLNOP and DmpLNOP, respectively (1,2). In an attempt to eliminate oxygenase component expression levels as a variable, two new plasmids, pPOW100 and pPOW101, were constructed such that transcription started at dmpL in both cases. This was done by replacing dmpP in pVI293 by dmpK to generate pPOW100, , respectively, and activity is expressed per milligram of DmpLNO. The assay procedure was as outlined under ''Experimental Procedures,'' except that DmpK was added to DmpLNO and this mixture was incubated on ice for several minutes before addition to the assay: however, premixing was not essential for observing inhibition. This data was obtained using DmpK purified from the first peak of the Phenyl-Sepharose column eluate, but similar results were obtained for other purified fractions of DmpK. and by deleting dmpP in pVI293 to generate pPOW101 (see ''Experimental Procedures''). Since DmpP is expressed at a much lower level in E. coli than the DmpLNO polypeptides (see Figs. 4 and 5 in Ref. 1), DmpK should be produced from the dmpP ribosome binding site in pPOW100 at a low level, which interferes minimally with in vitro hydroxylase activity measurements. Neither of the other two polypeptides required for optimal hydroxylase activity (DmpM and DmpP) are expressed in these constructs, but are provided in activity assays.
As can be seen in Fig. 4, expression of the polypeptides of the oxygenase component (cf. lane 7) from pPOW100 and pPOW101 is low in the absence of IPTG induction (lanes 1 and 2) and increases at 1.25 (lanes 3 and 4) and 3 h (lanes 5 and 6) post-induction, with essentially the same levels observed in both crude extracts. Note also the position of the DmpK band (lane 8), which is not distinguishable in the samples of crude extract (lanes 1-6), although it was readily detectable by Western blotting in extracts from cells harboring pPOW100 (data not shown). Based on these results, one would expect both crude extracts to exhibit the same levels of phenol hydroxylase activity in vitro, with some inhibition by DmpK possible in the extract from cells harboring pPOW100.
As is shown in Table II, this was not what was observed. In crude extracts obtained from the construct expressing DmpK (pPOW100), levels of oxygenase activity were high compared to extracts prepared from cells in which DmpK was absent (pPOW101). The possibility that extracts from DmpK-lacking cells contain some inhibitor was excluded by the observation that the oxygenase activity was additive in assays where both extracts were present (data not shown).
The addition of ferrous iron to the enzyme assay resulted in increased oxygenase activity for each extract (Table II), indicating that even when produced in the presence of DmpK not all of the oxygenase component was active. Since the levels of expression of the oxygenase component polypeptides appear to be essentially identical in extracts prepared from cells harboring the two plasmid constructs, it is interesting that the oxygenase activity was not activated to the same level in each case. In order to determine if the observed difference in ferrous iron stimulation was due to the presence or absence of DmpK, small amounts of purified DmpK were added to the in vitro assays. Dramatically, in the presence of added ferrous iron, inactive DmpLNO oxygenase expressed by cells harboring pPOW101 was activated by a small amount of purified DmpK. The level of activity achieved was almost the same as that of the oxygenase when co-expressed with DmpK in cells harboring pPOW100 (Table II). DISCUSSION While functional roles have been assigned to most of the 15 dmp operon-encoded proteins required for the mineralization of phenols by Pseudomonas sp. strain CF600, that of DmpK has remained obscure. Although the gene encoding this protein is clustered together with five phenol hydroxylase genes, its product is not essential for in vitro phenol hydroxylase activity. However, when the gene encoding this protein is deleted, strains that might normally be expected to be able to grow using multicomponent phenol hydroxylase fail to do so. These results led to the initial suggestion that DmpK might be involved in phenol binding/transport or phenol hydroxylase regulation (3).
In this paper, we have examined the interactions of DmpK with phenol hydroxylase in some detail. Since DmpK appears to be present at very low levels in crude extracts of phenolgrown cells, it was necessary to overexpress the protein in order to purify it. Very high levels of expression of this protein were attained from the T7 promoter in pVI203, and this made it possible to obtain large quantities of pure DmpK. Spectral characteristics of the purified protein indicated that it did not contain any common redox cofactors (e.g. FAD, heme, Fe-S centers), and iron assays indicated no significant quantities of iron. This latter observation was of interest, as some types of redox-active iron centers are spectroscopically invisible.
Purified DmpK was found to have a marked inhibitory effect on phenol hydroxylase activity when assays were performed in the presence of DmpM, an accessory protein required for optimal turnover of the hydroxylase. In the absence of DmpM, DmpK had little effect on activity. The simplest interpretation of this observation is that DmpK in some way interferes with the interaction between DmpM and the oxygenase component of the hydroxylase. This could be mediated by binding of DmpK either to the oxygenase component or to DmpM, such that it would interfere with the normal DmpM-oxygenase interaction.
In relatively dilute solutions of oxygenase component containing a severalfold excess of DmpK, it was found that DmpK could be cross-linked to both DmpL and DmpN, the two largest subunits of the oxygenase component. In contrast, when mixtures of DmpM and DmpK were exposed to various crosslinking reagents, no evidence of cross-linked products was found. Thus, while interactions between DmpM and DmpK cannot be ruled out, it is clear that DmpK interacts with oxygenase component on at least two of its subunits. It is interesting to note that we have also found DmpM to interact with at least one of the same subunits (DmpN). 3 These observations at least partially explain the inhibition of phenol hydroxylase activity in vitro. However, since DmpK is expressed at much lower levels than the polypeptides of the oxygenase component, inhibition of phenol hydroxylase is unlikely to be the main physiological function of this protein.
Results obtained using the two plasmid constructs, pPOW100 and pPOW101, are consistent with a role for DmpK in the formation of active oxygenase component. When the oxygenase component polypeptides were expressed in the absence of DmpK from pPOW101, no oxygenase activity was detected in crude extracts; expression in the presence of DmpK from pPOW100 led to the formation of active oxygenase. Although DmpK is inhibitory to phenol hydroxylase even when it is present at a less than molar ratio, the level of expression of DmpK from pPOW100 is apparently low enough relative to the oxygenase polypeptides so that inhibition is minimal. The low level of DmpK expression from pPOW100 is likely to be similar to the level at which DmpK is expressed in Pseudomonas sp. strain CF600. Therefore, activation of the oxygenase by DmpK in the recombinant strain probably reflects the physiological role of this protein.
Clues about the mechanism of oxygenase component activa-tion are provided by the results obtained when ferrous iron and purified DmpK were added to the hydroxylase assays (Table  II). The 2-fold increase in activity caused by adding iron suggests that crude extract from cells co-expressing DmpK and oxygenase contained some inactive oxygenase, probably apoenzyme. Possible explanations are that the level of DmpK coexpression was not optimal, or that some apoenzyme was produced during cell breakage. By contrast, in the absence of DmpK co-expression, all of the oxygenase was present in an inactive form, and addition of ferrous iron resulted in very little activation. However, when a slightly inhibitory concentration of DmpK was also present in the assay, activation in the presence of iron was dramatic for the inactive DmpLNO oxygenase generated in the absence of DmpK. These results strongly suggest a role for DmpK in post-translational insertion of iron into apo-oxygenase. In this context, it is interesting to note that cross-linking experiments revealed a direct interaction between DmpK and the oxygenase component subunit, DmpN, which encompasses the putative ligands for the binuclear iron center (3,5). Experiments using bacterial strains in which dmpK was deleted showed that DmpK is essential for allowing growth on phenol using the multicomponent phenol hydroxylase (1). The results reported here indicate a role for DmpK in post-translational incorporation of iron into the oxygenase component of the phenol hydroxylase. Since the oxygenase component was expressed in the absence of phenol, reductase (DmpP) and the activator protein (DmpM), it was incapable of turning over at any significant rate. This means that DmpK does not simply play a repair role for oxygenase, which might inadvertently lose iron via a turnover event, as oxygenases are sometimes prone to do; instead, it must be essential for proper de novo assembly of the active oxygenase. However, an ancillary role for DmpK in oxygenase repair is certainly possible.
Requirements for accessory proteins in the assembly of metalloproteins have previously been well documented for some proteins, including the nickel-containing enzyme, urease, and the FeMo protein of nitrogenase (reviewed in Refs. 27 and 28, respectively). Although the functions of some of these accessory proteins are not known, others appear to function as scaffold proteins for the assembly of a metal cofactor (NifEN (see Ref. 28, and references therein), potential metal donors (of iron for Nif U (29), and of nickel for UreE (30)), and chaperone or ''molecular prop'' proteins thought to be important in metalloprotein assembly or metal center incorporation (UreD (31) and NifY (32)). Like some of these proteins, DmpK appears to be able to function post-translationally. However, unlike NifEN or 3 E. Cadieux and J. Powlowski, unpublished observation. NifU, purified DmpK does not contain any iron, and unlike the scaffold protein, NifEN, DmpK exhibits no sequence similarity to the putative iron center-containing polypeptide (DmpN). Thus, although it is possible that DmpK loses an essential cofactor during isolation, the available evidence is consistent with a function as some sort of molecular prop required to allow correct or efficient incorporation of iron into the oxygenase component of phenol hydroxylase.
The outstanding questions about how DmpK functions are important ones, both in furthering our understanding of phenol hydroxylase, and in determining how best to express recombinant phenol hydroxylase for future studies of structure-function relationships. It is interesting to note that activator proteins like DmpK have not been reported previously for binuclear iron center oxygenases other than phenol hydroxylases, although a gene encoding a protein with some sequence similarities is associated with toluene monooxygenase genes. Considering the relatively recent discovery of enzymes like the multicomponent phenol hydroxylase, it may well be that proteins like DmpK are more widespread than is currently known.