Dimethoxyphenol oxidase activity of di¡erent microbial bluemulticopper proteins

Francisco Solano a, Patricia Lucas-El|o b, Daniel Lopez-Serrano a, Eva Fernandez a,Antonio Sanchez-Amat b;*

a Department of Biochemistry and Molecular Biology B, University of Murcia, Murcia 30100, Spainb Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, Murcia 30100, Spain

Received 24 April 2001; received in revised form 14 August 2001; accepted 15 August 2001

First published online 3 October 2001

Abstract

2,6-Dimethoxyphenol is a versatile substrate for Pyricularia oryzae laccase, PpoA from Marinomonas mediterranea, phenoxazinonesynthase from Streptomyces antibioticus and mammalian ceruloplasmin. In addition, in cellular extracts of microorganisms expressing otherblue multicopper proteins with no enzymatic activity previously described, such as Escherichia coli (copper resistance CueO), Pseudomonassyringae and Xanthomonas campestris (copper resistance CopA), Bacillus subtilis (sporulation protein CotA) and Saccharomyces cerevisiae(iron transporter Fet3p), laccase activity is detected under appropriate conditions. This oxidase activity can be spectrophotometricallyfollowed by the oxidation of 2,6-dimethoxyphenol. Specific staining after SDS^PAGE is also possible for some of these proteins. Thisdetection assay can facilitate the study of the multiple functions that such proteins seem to carry out in a variety of microorganisms. ß 2001Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Blue multicopper protein; Oxidase; Laccase; Copper tolerance protein

1. Introduction

Blue multicopper proteins are a family of proteins dis-tributed in a great number of organisms including bacte-ria, fungi, plants and mammals. They are involved in avariety of enzymatic and non-enzymatic functions. Theseproteins show four short regions with high similarity in theamino acidic sequences, that consist of histidine-rich mo-tifs that constitute the copper-binding domains (Fig. 1).The copper ions, according to their environment and spec-troscopic properties, are classi¢ed as copper types 1 (blue),2 (normal) and 3 (coupled pair) [1].

The last motif, located near the C-terminus of the poly-peptide chains, is involved in the binding of type 1 and 3copper ions. It contains the conserved tripeptide HCH.

The pair of histidines bind copper type 3, but the centralcysteine and another histidine located close to this stretchbind copper type 1. This copper is the only one bound tothe peptidic chain through a ligand distinct of histidineimidazole groups and its environment is likely involvedin the di¡erences in the structure/function relationshipsof these proteins.

The copper-binding domains constitute the catalytic ac-tive site in enzymatic multicopper oxidases, such as lac-cases [2,3], phenoxazinone synthase [4], ascorbate oxidase[5], bilirubin oxidase [6] and ceruloplasmin, the copperprotein found in the blood of higher animals [5,7]. Onthe other hand, there are a number of multicopper pro-teins sharing the four copper-binding domains but in-volved in perhaps non-enzymatic cellular functions, suchas conferring copper tolerance to microorganisms [8^10],iron uptake in yeast [11,12], and sporulation [13,14]. Othermulticopper proteins lacking known functions have beenrecently postulated from direct sequencing of genomes,such as the Aquifex aeolicus periplasmic cell division pro-tein [15].

Laccases (benzenediol :oxygen oxidoreductase, EC1.10.3.2) are the most numerous group of blue multicop-

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 4 0 0 - 1

* Corresponding author. Tel. : +34 (968) 36 49 55;Fax: +34 (968) 36 39 63.

E-mail address: antonio@um.es (A. Sanchez-Amat).

Abbreviations: DMP, 2,6-dimethoxyphenol;DMPO, 2,6-dimethoxyphenol oxidase; PPO, polyphenol oxidase

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per proteins. They show polyphenol oxidase (PPO) activ-ity, and are able to oxidize o- and p-quinols, aminophe-nols, phenylenediamines, anilines and benzenethiols, al-ways coupled to the reduction of oxygen to water [2,16].The speci¢city to phenolic substrates varies within thegroup. Simple diphenols, such as hydroquinone and cat-echol, are good substrates for most laccases, but the lowmolar adsorption coe¤cient at the visible region and theinstability of the quinonic products have limited their useas substrates for standard assays. Synthetic substrates withhigher molar visible absorption coe¤cient for the oxidizedproducts have been used in a number of published studies,such as o-dianisidine, ABTS (2,2P-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)) and syringaldazine (N,NP-bis(3,5-dimethoxy-4-hydroxybenzylidene hydrazine)) [2,6,17^20]. The corresponding oxidized chromogens fromthese substrates are not stable yet, and the assays usuallyneed a rather acidic optimal pH. Thus, methoxy substi-tuted phenols, such as guaiacol [21] or 2,6-dimethoxyphe-nol (DMP) [3,22^24], have also been used to follow theenzymatic activity of these copper oxidases.

The copper-binding sites of laccases are shared by otherblue multicopper proteins (Fig. 1). As most of these pro-teins have physiological roles apparently not related toenzymatic catalysis, it has been presumed that they donot show oxidase activity. The detection of these proteinshas been laborious, and frequently in vivo assays relatedto speci¢c functions have been employed. Copper toler-ance factors [8] have been quantitated by immunoblotwith antibodies to chimeric proteins obtained by fusionwith L-galactosidase. Iron transporters [11,12] have beenmeasured by membrane transport assays with radioactive59Fe. However, the line between enzymatic and non-enzy-matic blue multicopper proteins is not clear. The proteininvolved in Escherichia coli copper tolerance, CueO (yacKgene product) has been denominated recently Cu e¥uxoxidase [25]. Thus, the oxidase capacity of this proteinfamily should be reinvestigated to facilitate its detectionin microbial cells and the elucidation of their mechanismsof action and functions. This paper shows that these pro-teins exhibit a certain dimethoxyphenol oxidase (DMPO)activity under appropriate conditions, that allows for theirquick detection.

2. Materials and methods

2.1. Microbial strains, culture and cell extract preparation

Marinomonas mediterranea (CECT 4803, ATCC700492) was cultured at 25³C in a liquid complex marinemedium, MMC [26]. E. coli K-12 strain MG1655 wascultured at 37³C, Pseudomonas syringae ATCC 19304and Xanthomonas campestris ATCC 33913 were grownat 25³C, all of them on LB agar plates. For extractionand expression studies of blue multicopper proteins, thebacteria were inoculated in liquid LB medium supple-mented with 0.25 mM copper sulfate, unless otherwisespeci¢ed in particular experiments.

Bacillus subtilis ATCC 6051 was grown on the manga-nese-containing nutrient agar sporulation medium [27].Sporulation was induced as described [13]. Essentially,cells were allowed to grow overnight on the surface of aplate until a barely visible lawn appeared. Lastly, cellswere scraped o¡ the plate and suspended in the abovementioned liquid sporulation medium. At di¡erent timeintervals, aliquots were removed and used to determineDMPO activity. Streptomyces antibioticus ATCC 8663was grown in YEME medium at 25³C until the micro-organism reached the stationary phase of growth. Finally,Saccharomyces cerevisiae X2180-1A was grown in YPDmedium at 28³C supplemented with 0.2 mM bathophenan-throline to induce iron deprivation [11].

To collect the cells, 25 ml of the di¡erent cultures werecentrifuged at 5000Ug at 4³C for 6 min. In the case of theStreptomyces, the mycelia were collected by ¢ltration. Thecellular pellets or the mycelia were washed once with iso-tonic solution and centrifuged again in the same condi-tions. The pellets were then resuspended in 1 ml of 0.1 Msodium phosphate bu¡er pH 5.0 (the same bu¡er used forenzymatic assay, see later), pipetted in an Eppendorf tubeand disrupted by sonication using a Braun Labsonic U(4 min treatments at a relative output power of 0.5 with0.7 s of duty periods). Yeast cells were disrupted by vor-texing during 12 min in the presence of glass beads.

As some of the blue multicopper proteins are hydro-phobic membrane proteins (i.e. Fet3p, PpoA), to obtaintotal solubilized extracts the sonicated homogenates were

Fig. 1. Alignment of copper-binding domains in blue multicopper proteins. The amino acidic length of every protein (Aa), accession numbers to publicdatabases (GB for GenBank and SP for Swiss Prot) and the four conserved metal-binding residues for copper types 1, 2 and 3 are shown (shadowbackground). Central line separates prokaryotic from eukaryotic proteins. The consensus for copper ligands is shown in the bottom line.

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treated with a non-ionic detergent such as 1% Igepal CA-630 [28] for 15 min and then centrifuged at 105 000Ug at4³C for 30 min in a TL-100 Beckman ultracentrifuge toobtain the solubilized total protein extract.

2.2. Enzymatic determinations

DMPO activity was determined for cell extracts or so-lutions of commercial enzymes: laccase (from Pyriculariaoryzae, 389 units/mg) and bovine ceruloplasmin (3000units/ml) from Sigma Chem. Co. (St. Louis, MO, USA).DMPO activity was spectrophotometrically determined byrecording the increase in optical density at 468 nm. Theoptical density coe¤cient of the quinonic oxidized prod-uct, 3,3P,5,5P-tetramethoxydiphenylquinone, is 14 800 M31

cm31 [29]. The assay mixture consisted of 2 mM DMP in0.1 M sodium phosphate bu¡er, pH 5.0 and 0.1 mMCuSO4 when appropriate.

Analytical SDS^PAGE was performed as described byLaemmli [30], using an acrylamide concentration of 10%for the separating gel and 3% for the stacking gel. Resolv-ing bu¡er was Tris^HCl, pH 8.8, and reservoir bu¡erTris^glycine, pH 8.3, both containing 0.1% SDS. Sampleswere mixed in a ratio 2:1 (v/v) with sample bu¡er (0.18 MTris^HCl, pH 6.8, 15% glycerol, 0.075% bromophenolblue, 3% SDS). Electrophoresis was run at 20³C, and at15 mA for 20 min and 25 mA for about 1 h. Molecularmass calibration was performed using the prestained SDS^PAGE standards from Bio-Rad (low range). 2-Mercapto-ethanol was omitted from the sample bu¡er for theDMPO activity staining of gels. To perform the stain,the pH of the slabs were equilibrated at pH 5 by immer-sion in 0.1 M sodium phosphate, pH 5.0 for 5 min atroom temperature. Immediately, stain was carried out byincubating the gels in a freshly prepared solution of 2.5mM DMP and 0.2 mM CuSO4 in 20 mM sodium phos-phate, pH 5.0 at 37³C for at least 10 min. The stability ofthe staining allowed that the gels could be dried andscanned.

3. Results

Comparative experiments using di¡erent laccase sub-strates demonstrated that DMP was the most suitable sub-strate for this type of enzymatic assays as judged by anumber of factors including the stability of the coloredproduct, its high absorption molar coe¤cient, weak acidicoptimal pH and oxidation e¤ciency for a number of bluemulticopper enzymes. In some cases, such as laccase fromP. oryzae and bovine ceruloplasmin, it was possible to usethe puri¢ed enzyme, but for other cellular extracts con-taining blue multicopper oxidases were employed (see Sec-tion 2).

Alternative substrates, such as ABTS, were bettersubstrates for particular enzymes, such as laccase from

P. oryzae, but there were no substrate at all for othertested multicopper oxidases. In addition, ABTS needs arather acidic pH (around pH 3^4) for optimal enzyme-mediated oxidation. Overall, DMP was the most versatilesubstrate for designing a general enzymatic assay for de-tecting blue multicopper proteins.

The kinetics of the catalysis and the conditions of theDMPO assay suggested classifying the blue multicopperproteins in three groups. The ¢rst group contained laccasefrom P. oryzae, PpoA from M. mediterranea [3,28], phe-noxazinone synthase from S. antibioticus [4] and mamma-lian serum ceruloplasmin [7]. These proteins were able tooxidize DMP rapidly without copper supplementation andthey were characterized by the linear accumulation of theDMP oxidized product (Fig. 2A). This behavior should beconsidered as real laccase activity.

Other blue multicopper proteins were unable to oxidizeDMP under the conditions used for the ¢rst group. How-

Fig. 2. Time course of DMP (2 mM) oxidation by several blue multi-copper proteins in 0.1 M sodium phosphate at pH 5. The oxidation wasfollowed by an increase in the optical density at 468 nm. (A) P. oryzaelaccase (R, 10 Wg of the commercial available preparation); bovine plas-ma ceruloplasmin (E, 15 units of the commercial available preparation);PpoA from M. mediterranea (a) ; phenoxazinone synthase from S. anti-bioticus (F) ; CueO from E. coli (8) ; CopA from P. syringae (U) ;X. campestris (+). (B) DMPO for the Fet3p from S. cerevisiae (F) andCotA from B. subtilis (R). Observe that the last two reactions werevery slow and had to be followed for up to 24 h at 30³C. The appropri-ate volumes of solubilized crude microbial extracts containing 150 Wg ofprotein were used in all cases but the commercial available P. oryzaelaccase and ceruloplasmin. Supplementation with 0.1 mM CuSO4 wasneeded to observe DMP oxidation by extracts of the last ¢ve strains.

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ever, after copper supplementation in the reaction media,some of the tested proteins catalyzed a rapid DMP oxida-tion, as judged by the development of a yellowish colorafter addition of that ion. Hence, the assay conditions forDMPO activity were modi¢ed by adding 0.1 mM coppersulfate to the assay media. This group of blue multicopperproteins probably includes the copper tolerance proteinsCueO from E. coli, CopA from P. syringae and X. cam-pestris, as these proteins are contained in the respectivecellular extracts used. They underwent a burst of activityand later rapid inactivation as judged by the stop in prod-uct accumulation observed after a few minutes (Fig. 2A).As this activity was dependent on the addition of CuSO4

to the reaction assay, it was considered as pseudo-laccase,but it maybe helpful for blue multicopper protein detec-tion yet.

Finally, other blue multicopper proteins, possibly theFet3p yeast membrane protein involved in iron transport,and the CotA sporulation protein from B. subtilis, showeda very slow DMP oxidation rate (Fig. 2B). These assayslasted hours in the presence of copper, and DMP oxida-tion was not detectable in its absence.

As some puri¢ed blue multicopper proteins, but alsocellular extracts containing some of these proteins wereused, the characterization of the DMPO activity depen-dent on copper addition to these proteins was furtherstudied. A series of controls were performed to character-ize the assay speci¢city related to the protein and the metalion. First, addition of CuSO4 up to 0.25 mM in the ab-sence of proteins, or to solutions of other proteins, such asbovine serum albumin or catalase, did not catalyze DMPoxidation in the conditions of the assay for at least 3 h(Fig. 3A). Second, boiling of the bacterial extracts contain-ing the blue multicopper proteins eliminated copper-de-pendent activation. Third, at the same concentration asCu2�, other sulfate salts of cations, such as Ni2�, Co2�

or Zn2�, failed to produce DMPO activity in the condi-tions of the assay. Finally, total extracts of some microbialcultures, such as non-sporulating B. subtilis, were unableto catalyze DMP oxidation even in the presence of copperions. Overall, all these negative controls strongly suggestthat the DMPO activity measured should be related to theinteraction of the copper added to the cellular extractswith the blue multicopper proteins presumably containedin those extracts.

The DMPO activity of these proteins depends on copperin a saturable manner up to 100^200 WM (Fig. 3A). In ourhands, higher copper concentrations could not be assayedbecause of precipitation of copper phosphate under theconditions of the assay. This side e¡ect was also observedin the blank controls, making the spectrophotometric mea-surements di¤cult. Interestingly, during the revision ofthis work, a recent publication describes the use of amuch higher copper concentration in acetate bu¡er [31].In agreement with our results, a saturated similar pro¢le isalso found for the CueO from E. coli.

Some additional experiments were performed to supportthe correlation between the DMPO activity observed withthe induction of the blue multicopper proteins in microbialextracts. A transcriptional activation of cueO by copperhas been described [25], so that it is reasonable to assumethat the protein levels would also rise in response to cop-per. In the same way, copper tolerance proteins are in-duced by copper excess [8]. Thus, we studied the responseof DMPO activity in cellular extracts of microbial culturesgrown in di¡erent concentrations of copper sulfate. Theinduction of DMPO activity in E. coli extracts from cul-tures grown at increasing concentrations of CuSO4 up to1 mM was observed (Fig. 3B). Similar response curves tothe addition of copper were obtained for P. syringae andX. campestris. In both cases, CuSO4 addition to culturemedia caused an approx. 150^200% increase in the ob-served DMPO activity (data not shown).

B. subtilis CotA is involved in sporulation [13,14]. Thisprotein seems to also be responsible for the slow oxidationof DMP in our assay conditions, as we were unable todetect this activity in non-sporulating cultures or those

Fig. 3. (A) E¡ect of the amount of Cu2� added to the assay media onthe DMP oxidation by a control bu¡er solution (+) and crude extractsfrom E. coli (b), P. syringae (F) and X. campestris (R). All assays werecarried out at pH 5 using 50 Wl of extract containing approx. 3 mg/mlof protein. (B) Induction of CueO, determined as DMPO activity, inE. coli by di¡erent concentrations of Cu2� added to the culture medium(LB). These data are the mean of two di¡erent sets of induction experi-ments. A similar induction pattern was observed for the copper toler-ance CopA proteins from P. syringae and X. campestris.

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with a low percentage of sporulation. The addition ofcopper up to 1 mM to cultures of B. subtilis also failedto induce DMPO activity.

DMPO was also assayed for blue multicopper proteindetection after SDS^PAGE by designing a speci¢c stainmethod of these proteins. After electrophoresis in the ab-sence of 2-mercaptoethanol, the presumable bands of thecopper tolerance proteins, CopA from X. campestris andP. syringae, and CueO from E. coli, could be visualizedwhen CuSO4 was added to the gel slab (Fig. 4). The cal-culated apparent molecular masses corresponding to eachband, respectively 67, 64 and 57 kDa for the respectiveblue multicopper proteins from X. campestris, P. syringaeand E. coli, are in the range deduced from gene sequencesfor their respective polypeptidic chains, 592, 577 and 516amino acids, once the respective putative signal peptide ofthese proteins is subtracted. These results strongly indicatethat the DMPO bands stained were due to the appropriateblue multicopper proteins contained by the bacterial ex-tracts. However, staining for the Fet3p from yeast and theCotA from B. subtilis was not achieved, very likely due tothe low DMPO activity of these proteins.

4. Discussion

Blue multicopper proteins have been assigned to verydi¡erent biological functions. The most studied proteinsof this group are fungal laccases because of their PPOactivity and biotechnological applications. These enzymes

are able to oxidize a number of phenolic substrates medi-ated by a copper-containing active site. This enzymaticactivity has been suggested for other blue multicopperproteins [32] due to the high similarity in the amino acidmotifs of copper-binding sites (Fig. 1), but experimentalevidence is lacking yet. Thus, we explored whether theseproteins express some laccase activity under some generalappropriate conditions.

A number of substrates have been used for detectingthis activity [2,3,15,21^24], but our data indicate thatDMP is the best choice because of the wide capacity ofoxidation and the quite high absorption molar coe¤cientand stability of its oxidized dimeric colored form,3,3P,5,5P-tetramethoxydiphenylquinone. The coe¤cient ofthis quinone at 468 nm is 14 800 M31 cm31, almost threetimes higher than the one obtained by oxidation of o-me-thoxyphenol (guaiacol) to tetraguaiacol (5570 M31 cm31)[29]. In this work, it has been shown that all the bluemulticopper proteins tested have indeed some DMPO ac-tivity that can be determined spectrophotometrically atpH 5. In addition, some of these proteins can also be vi-sualized by speci¢c gel staining after SDS^PAGE. Otherpossible substrates such as o-dianisidine, ABTS and syrin-galdazine were tested, but they were discarded because ofthe inability to be oxidized by some of these proteins. Inaddition, syringaldazine and ABTS need neutral andrather acidic pH, respectively. At neutral pH, seriousproblems due to spontaneous oxidation of the substrateby copper ion occur when this ion is added. At pH around3, optimal for ABTS, the maintenance of the structure ofsome blue multicopper proteins would be compromised.DMPO can be assayed at pH 5.0, which circumventsboth problems. Moreover, the stability of the oxidizedcolored product assures that it can be revealed by gelstaining after SDS^PAGE under appropriate conditions.

Di¡erent laccases and non-laccase blue multicopperproteins were assayed but not all of them showed thesame e¤ciency to catalyze the DMP oxidation. In the lightof the obtained kinetic pattern, three possible groupscould be established. Authentic laccase from P. oryzae,M. mediterranea PpoA, phenoxazinone synthase and ce-ruloplasmin belong to the ¢rst one. This group is charac-terized by a time course linear activity. These proteins arenot inactivated during the catalysis, and they do not needCu2� supplementation to exhibit DMPO activity.

The second group, containing the bacterial copper tol-erance factors, is dependent on the addition of Cu2� toexhibit DMPO activity. They show a burst in its initialactivity, but they are rapidly inactivated. This behavioris considered a pseudo-laccase activity. It is quite likelythat the activity appearance is due to the binding ofCu2� to some of the copper-binding centers and concom-itantly expression of the oxidase activity, but the use ofcrude extracts rather than puri¢ed proteins made this as-sumption of direct interaction between copper and bluemulticopper protein premature. On the other hand, other

Fig. 4. Electrophoretic analysis of the DMPO activity in X. campestris,P. syringae and E. coli. 10% SDS^PAGE were run under dissociatingconditions in the absence of 2-mercaptoethanol and stained at 37³Cwith a solution of 2.5 mM DMP in 20 mM sodium phosphate pH 5.0and 0.2 mM of copper sulfate. Prestained molecular markers (lane 1)were from Bio-Rad.

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indirect data support a correlation between DMPO activ-ity and the blue multicopper protein levels. It has beenshown that its level is increased in response to the additionof copper to the culture medium (Fig. 3B), in agreementwith recent reports on the transcriptional activation of theyacK gene encoding CueO [25]. Although the induction ofalternative unknown proteins di¡erent from the coppertolerance proteins cannot be completely ruled out, the re-sults obtained provide further support to the correlationbetween DMPO activity and the levels of blue multicopperproteins in microbial extracts. Further studies on the rela-tionships between the enzymatic DMPO oxidation and itsphysiological role in copper and iron homeostasis wouldbe interesting. In this regard, a role in the oxidation ofphenolate siderophores by CueO has been very recentlyreported [31].

Finally, the third putative group is probably representedby the yeast iron transport protein, and the CotA sporu-lation protein from B. subtilis. These proteins also show aDMPO activity dependent on Cu2� supplementation. Inaddition, and opposite to the former group, the oxidationis very slow but it does not undergo inactivation. Thus,the detection assay requires long incubation periods forthese proteins. The di¡erent pattern of DMPO activitybetween the second and third group could be related todi¡erences at the copper-binding sites of those presumablynon-enzymatic blue multicopper proteins in comparison tolaccases. In fact, signi¢cant di¡erences can be seen in thethird region of the copper-binding motif of CotA (Fig. 1).Other uncharacterized factors would account for the cata-lytic di¡erences among the proteins herein studied. TheDMPO activity due to the B. subtilis CotA protein wasonly detectable in sporulating cultures, and it was not in-ducible by copper, in agreement with its proposed role inthe formation of the spore coat [13].

In conclusion, the DMPO assay presented in this papercan facilitate the detection, quantitation and further char-acterization of blue multicopper proteins, o¡ering a con-venient tool to detect actual levels of these proteins, evenin crude microbial cellular extracts.

Acknowledgements

This work was supported by grant PB97/1060 from theCICYT (Spain). P.L.-E. is grateful to the Seneca Founda-tion (Murcia Region) for a predoctoral fellowship to par-ticipate in this project. E.F. was a recipient of a fellowshipfrom the Ministerio de Educacion y Cultura, Madrid. Theauthors are grateful to Drs. T. Soto and G. Plunkett IIIfor providing some microbial strains.

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