A new multicopper oxidase from Gram-positive bacterium Rhodococcus erythropolis with activity modulating methionine rich tail

Protein Expression and Purification 89 (2013) 97–108

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A new multicopper oxidase from Gram-positive bacterium Rhodococcus erythropolis with activity modulating methionine rich tail Thomas Classen ⇑, Jörg Pietruszka ⇑, Saskia Marina Schuback Insitut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, D-52426 Jülich, Germany

a r t i c l e

i n f o

Article history: Received 13 December 2012 and in revised form 6 February 2013 Available online 24 February 2013 Keywords: Multicopper oxidase Cuprous oxidase Bacterial laccase Expression of holo-form

s u m m a r y Multicopper oxidases are involved in a wide variety of physiological tasks in nature. They are part of the lignin formation/decomposition system in plants and fungi. In bacteria they are part of developmental processes and the heavy metal resistance apparatus. A well characterised example is the copper tolerance protein CueO of Escherichia coli (CueOEC). Here, we report the heterologous expression of the apo- and holo-form of CueORE, a homologue to CueOEC from Rhodococcus erythropolis. Upon incubation with copper(II) ions, low active apo-CueORE was converted into the active holo-CueORE in vivo. The holo-form was physico-chemically characterised using a copper(I) BCA complex and the model substrate 2,6-dimethoxyphenol. The spectroscopic and catalytic properties are different from CueOEC, revealing a high catalytic efficiency (kcat/Km) of 115 min1 mM1 with physiological Km of 80 lM for the cuprous oxidase activity. At the C-terminus of CueORE a methionine rich tail region was identified which can be found in a variety of actinobacteria. Chimeras of the E. coli and R. erythropolis enzymes were constructed to investigate the influence of this tail regarding kinetic parameters. It was shown that the tail did not have the same function as the corresponding methionine rich loop in CueOEC. However, it modulated the kinetic properties of the enzyme. Ó 2013 Elsevier Inc. All rights reserved.

Introduction Multicopper oxidases (EC 1.16.3.x, 1.10.3.x, and 1.7.2.x) exhibit a broad substrate spectrum and fulfil many physiological functions in nature. They almost ubiquitously occur in fungi, higher plants, bacteria, and insects and are involved in lignin formation/degradation, cell development, or heavy metal resistance [1–6]. As biocatalysts, multicopper oxidases are able to reduce molecular dioxygen to water and use the necessary four electrons origin from the coupled oxidation of nitrite, ascorbate, copper, or from the oxidative C–C coupling in case of laccases and bilirubin-oxidases [6–16]. To date, our understanding mainly originates from fungal enzymes. However, the knowledge of bacterial multicopper oxidases has been enormously extended in the past ten years [17–19]. Due to the economically and ecologically sustainable oxidation agent dioxygen, without the need of expensive co-factors, multicopper oxidases find applications in several biotechnological processes like dye decolouration, delignification of wooden fibres, bioremediation, polymer synthesis or food processing [12,20]. Both, fungal and bacterial multicopper oxidases, contain four copper ions: One type I (T1) copper, which abstracts the electron ⇑ Corresponding author. Fax: +49 (0)2461 61 6196. E-mail address: [email protected] (J. Pietruszka). 1046-5928/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.02.003

of the substrate, one type II (T2), and two type III (T3) centres. The T3centresform a trinuclear core with a hydroxyl anion and the T2 copper in proximity. This trinuclear cluster deals with the storage and transfer of electrons to molecular oxygen, which is thereby reduced to water [21,22]. The blue colour of the blue copper enzymes originates from the complexed T1 copper (cf. Fig. 1A). Apart from catalysing oxidation reactions, the well characterised cuprous oxidase CueOEC is involved in the intrinsic copper resistance apparatus of Escherichia coli [11,23–30]. It mediates copper resistance via two modes of action: First, cytosolically expressed CueOEC stably binds four copper ions upon folding. The protein is then exported to the periplasmatic space via the twin arginine translocase pathway. Second, the enzyme then oxidises copper(I) to less toxic copper(II), which also prevents reuptake of copper into the cell. As a special feature, CueOEC has an additional, fifth, labile copper binding site (type IV, T4), which regulates the T1 copper site and is able to bind and oxidise copper(I) [26] (cf. Fig. 1B). Although, so far only fungal laccases are of industrial relevance, biotechnological applications of bacterial laccases would be favourable due to faster host growth, a higher yield of expressed proteins, a lower sensitivity towards halides and alkaline conditions, and enhanced stability of spore coat laccases such as CotA from Bacillus subtilis [31]. In addition, bacterial laccases do not

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Fig. 1. Mechanism of CueOEC as cuprous oxidase. (A) Molecular mechanism of CueOEC illustrated by the particular copper centres (orange circles). Copper(I) is bound labile at the T4 centre and oxidised to copper(II). The abstracted electron is transferred to the T1 and then to the T3 and T4 centres, where the oxygen reduction takes places. The picture was generated from pdb:1N68. (B) Schematic cut through an E. coli cell to illustrate the two modes of action in copper detoxification. CueOEC detoxifies the cytosol from copper(I) by binding four ions and its consequent export within the protein. Once reached the periplasmic space, CueOEC works as a cuprous oxidase coverting Cu(I) to the less toxic Cu(II).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

exhibit the diverse glycosylation pattern known from fungal enzymes and therefore should simplify heterologous expression and genetic engineering, e.g., to tailor the low substrate specificity for use in fine chemical synthesis. In the present study, we characterised the first known multicopper oxidase CueORE from Rhodococcus erythropolis DSM311. The ubiquitous existence in nearly every habitat -from salt water to soils- combined with public access to the sequences of the large genome make Rhodococcus a ‘warehouse’ to screen for new enzymes [32–34]. The biocatalytic potential of R. erythropolis was already realised in 1928 when Thornton and Gray reported aromatic compound decomposition by soil bacteria including Rhodococci, a reaction that is very challenging to many other organisms [35]. Experimental section Materials Chemicals were purchased from Carl Roth, Karlsruhe, Germany, and Sigma–Aldrich, Steinheim, Germany in analytical grade. For cloning, enzymes were bought from Fermentas, St. Leon-Roth, Germany, the vector pET22b was purchased from Merck, Darmstadt, Germany, and oligonucleo-tides were obtained as desalted lyophilisates from Sigma–Aldrich, Steinheim, Germany. R. erythropolis DSM311 (ATCC 15592) was acquired from DSMZ, Braunschweig, Germany as dry culture. Chromatographic columns and matrices were purchased from GE Healthcare, Munich, Germany, and Qiagen, Hilden, Germany, respectively. Chromatographic steps were carried out on ÄKTA purifier from GE Healthcare, Munich, Germany. For cell disruption, a French pressure cell press from Thermo Fisher Scientific, Schwerte, Germany, was used. The gene of CueOEC:MetRE was ordered synthetically from GenScript (Piscataway, NJ, USA). Cultivation of R. erythropolis DSM311 R. erythropolis DSM3111 was cultivated at 26 °C in tryptic soy broth (1.7% tryptone/peptone pancreatic digest from casein, 0.3% 1 Nucleic acid sequence of recombinant CueORE from R. erythropolis DSM311 was deposited under GenBank ID 1474724. The genomic fragment was deposited under 1512579.

soy peptone, 0.5% sodium chloride, 0.25% K2HPO4, pH 7.3, 0.25% Dglucose [all in w/v]) as described [35,36]. Solid media contained 1.5% [w/v] agar.

Cloning All constructs in this work (Fig. 6) are based on a modified pET22b-vector bearing a sequence coding for a N-terminal hexahistidine tag followed by a three amino acid linker (AMT), a TEVprotease recognition site (ENLYFQG), and restriction endonuclease sites NdeI, NcoI and XhoI. This vector is referred to as pHT in the following. The oligonucleotides used in this work are listed in the Supplemental information. The full length gene for cytosolic expression of CueORE was amplified from R. erythropolis DSM311 cells by colony PCR using Pfu-polymerase and the oligonucleotides RE_NcoI_fw and RE_XhoI_rv. Variants of CueORE were constructed as follows: Deletion of the last 21 C-terminal amino acids leading to pHT:cueOREDMetRE was achieved following the method of Tillet et al. [37] using the oligonucleic acids RE_DMetRE_fw, RE_DMetRE_fw_tlß RE_DMetRE_rv and RE_DMetRE_rv_tl. The sequence coding for the methionine rich loop of CueOEC was inserted into vector pHT:cueOREDMetRE by using the MEGAWHOP method of Miyazaki et al. [38]. The megaprimer was formed via PCR from E. coli DH5a genome using the oligonucleotides RE_MetEC_fw and RE_MetEC_rv, the product was used as double stranded oligonucleotides onto pHT:cueOREDMetRE to form pHT:cueORE:MetECDMetRE. The construct CueOEC:MetRE was ordered as synthetic gene from GenScript (Piscataway, NJ, USA) in proprietary pUC57-vector and finally cloned into pHT via NcoI and XhoI. Derivatives of CueOEC were constructed as follows: Deletion of the methionine rich loop of CueOEC (MetEC) leading to pUC57:cueOECDMetEC:MetRE was achieved by following the method of Moore et al.[39–41], named round-the-horn PCR, using the oligonucleic acids EC_DMetEC_fw and EC_DMetEC_rv, both 50 -phosphorylated, on pUC57:cueOEC:MetRE. The vectors pUC57:cueOEC and pUC57:cueOECDMetEC were created by round-the-horn PCR using the oligonucleotides EC_DMetRE_fw and EC_DMetRE_rv on pUC57:cueOEC:MetRE and pUC57:cueOECDMetEC:MetRE as template, respectively. The genes of interest in the pUC constructs were cloned into the pHT vector

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to obtain the vectors pHT:cueOECDMetEC:MetRE, pHT:cueOEC and pHT:cueOECDMetEC. Colorimetric copper determination assay According to Djoko et al. [23] a complexometric assay with bathocuproinedisulfonic acid (BCS) was applied. Copper(I) forms a purple complex with BCS, while copper(II) does not. A reduction of copper(II) to copper (I) can thereby detected colorimetrically. As a modification to the present procedure, urea was used instead of guanidinium hydrochloride as chaotropic reagent to prevent precipitation of the dye. To 950 lL of assay buffer (8 M urea, 300 lM BCS, 10 mM ascorbic acid) 50 lL of test solution (e.g. culture supernatant) were added, vigorously mixed and boiled for 10 min, because the presence of protein decreased the formation rate of the dye complex formation (data not shown). After cooling to ambient temperature the absorption at 482 nm was measured and compared to an identically treated reference sample series containing up to 100 lM copper(II)sulphate in aqueous solution. Expression of CueORE/EC Cytosolic expression of apo-CueORE was carried out in a 3 L baffled Fernbach-flask using 1 L terrific broth (TB) with 100 lg/mL ampicillin inoculated by 1% (v/v) of an overnight culture of E. coli BL21(DE3) pHT:cueORE grown at 37 °C. After incubation at 25 °C and 120 rpm for 9 h, the expression was induced with 0.1 mM IPTG followed by further incubation for 15 h. Holo-CueORE was expressed cytosolically following the protocol for apo-CueORE but using TB medium supplemented with copper(II)sulphate. For the expression tests, the medium was supplied with different concentrations of copper(II)sulphate (0–50 mM) and incubated over night at ambient temperature to equilibrate the system. Before inoculation, a 1 mL sample was taken, while the remaining medium was inoculated and treated as described above. After cultivation another 1 mL sample was taken. The samples were centrifuged at 18 k rcf (relative centrifugal force) for 20 min and the copper content in the supernatants was determined as described above. The cells were harvested and holo-CueORE was purified using immobilised metal chelate affinity chromatography (IMAC; vide infra). Finally, all mutants of CueORE and CueOEC were expressed as holo-form in 1 L TB medium supplemented with 10 mM copper(II)sulphate at 25 °C in a 3 L Fernbach-flask (inoculum with 1% over night culture, induction with 0.1 mM IPTG after 10 h, harvest after 24 h). Purification of CueORE For purification of apo-CueORE and all holo-CueOEC mutants, E. coli BL21 (DE3) cells from expression were resuspended (20% [w/v]) in buffer P0 (100 mM TRIS/HCl pH 8) and disrupted via French pressure cell press. After centrifugation at 4 °C and 18 k rcf for 20 min, the cleared lysate was loaded onto a 5 mL Qiagen NiNTA Superflow cartridge (equilibrated with P0) for IMAC. The column was washed with 5 column volumes (CV) P0 and 6 CV P30 (P0 with 30 mM imidazole). Finally, the protein was eluted with 6 CV buffer P250 (P0 with 250 mM imidazole) and concentrated to 10 mg/mL using an ultrafiltration unit with a molecular weight cut-off (MWCO) of 10 kDa (Sartorius Stedim Biotech, France). The protein solution was desalted over a PD10 column equilibrated with P0. The final eluate was clear and colourless. All holo-CueORE mutants were purified using the same protocol as for apo-CueORE, except that the stringent washing step with P30 buffer was omitted, because this fraction showed the same purity

99

as the eluted protein at 250 mM imidazole. The final protein eluate was of intense blue colour. To check the influence of the buffer composition on the protein, holo-CueORE protein was purified using the described purification protocol but using 100 mM 3-morpholinopropane-1-sulfonic acid/NaOH (MOPS), 100 mM TRIS/HCl and 100 mM potassium phosphate (KPi) at pH 8, respectively. To determine the enzyme activity the standard 2,20 -azino-bis(3-ethylbenzthiazoline)-6-sulphonic acid (ABTS) assay was used (vide infra). Reconstitution of holo-CueORE To form holo-CueORE in vitro, purified apo-CueORE was incubated with different molar equivalents of copper(II) ions. Therefore, 0, 5, 10 and 50 molar equivalents of copper(II)sulphate were added to 2 mL of apo-CueORE (2.9 mg protein/mL = 50 lM). Samples were prepared with and without 1 mM dithiothreitol (DTT). Finally, the mixture was filled up to 2.5 mL with P0 and incubated over night at 5 °C. Subsequently, unbound copper and DTT were removed by PD10 desalting columns equilibrated with P0. Copper concentrations were determined as explained above and ABTS oxidation rates were recorded via enzyme kinetics (vide infra). Size exclusion chromatography (SEC) Sample and reference protein solutions (0.5 mL of 1 mg/mL each: dextran blue 2 MDa, sweet potato b-amylase 220 kDa, yeast alcohol dehydrogenase 150 kDa, bovine serum albumin 66 kDa, bovine erythrocytes carbonic anhydrase 29 kDa, horse heart cytochrome C 12.4 kDa, tryptophan 0.2 kDa) were applied to a HiLoad™16/60 Superdex™ 200 pg column equilibrated with P0 buffer and eluted isocratically with P0 buffer. The retention volume was determined from elution profiles showing absorption at 280 nm. The molecular size of CueORE was estimated from the calibration function of reference proteins by exponential regression. Enzyme assays Enzyme activity kinetics were measured in duplicates using a spectrophotometer (UV-1800, Shimadzu, Duisburg, Germany) equipped with a peltier heater/cooler cuvette mover (CPS-240A). As standard assay for testing protein stability, the oxidation of ABTS to the corresponding radical was used. The assay was carried out in 100 mM sodium acetate buffer pH 5.5 at 30 °C with 5 mM ABTS as substrate. The increase of absorbance was determined time-dependently at 420 nm e420 = 36,000 M1 cm1 [28]. Steady state kinetics were taken by varying the ABTS concentration in the assay from 0–100 mM. For determination of the pH-optimum, the acetate buffer was substituted by 100 mM KPi-buffer with pH 4, 5.1, 6, 7.5, and 8.5, respectively. To test the pH-stability, the elution buffer after enzyme purification (100 mM TRIS/HCl pH 8) was exchanged to 100 mM KPi pH 4–12.2 by PD10 desalting columns before storing the enzyme solution at 25 °C for four weeks. The temperature dependent activity was determined by varying the cuvette holder’s temperature from 25–70 °C and pre-incubation of the ABTS standard assay mix without enzyme. Temperature stability tests were carried out applying the ABTS standard assay using enzyme samples that were constantly preincubated in aliquots of 50 lL at defined temperatures from 35– 75 °C in a Biometra TProfessional basic gradient thermocycler (Göttingen, Germany). To check whether acetonitrile is a suitable co-solvent for laccase and cuprous oxidase activity tests, the stability of CueORE in the presence of 50% (v/v) acetonitrile in standard buffer was tested

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by incubation of the enzyme in this solution at 4 °C. A half-life of 3300 ± 345 min at 4 °C (data shown in Supplemental information) allowed neglecting the effect of acetonitrile born protein denaturation during the enzyme assays, which usually took 1 min. For all above-mentioned measurements, the standard ABTS assay or slight variations thereof were used to determine both the initial activity and the time-dependent activity. All data were fitted on y = y0 + A  ex/t using least square algorithm. The term tln(2) equals the half-life of the protein at each given condition. Steady-state kinetics for the laccase activity were determined using 2,6-dimethoxyphenol (2,6-DMP) as substrate dissolved in acetonitrile. The photometric assay was carried out in 100 mM sodium acetate buffer pH 5.5. The substrate concentration was varied between 0.025–50 mM, while the acetonitrile content of the assay mixture was kept constantly at 30% (v/v). Product formation was recorded at 477 nm (e477 = 14,800 M1 cm1, [28]). Cuprous oxidase activity was determined using copper(I) bicinchoninic acid (BCA) complex. The substrate copper(I)[BCA]2 was formed in situ from tetrakis(acetonitrile)copper(I) hexafluorophosphate [Cu(MeCN)4PF6], and 2.5 equivalents of bicinchoninic acid disodium salt. To prevent oxidation prior to use, Cu(MeCN)4PF6 was dissolved in degassed and argon flushed acetonitrile. However, the reaction buffer (100 mM sodium acetate buffer pH 5.5) was not degassed. The acetonitrile content was kept constant at 20% (v/v) for all measurements. The decrease of absorbance at 30 °C was recorded at 482 nm. The molar extinction coefficients of Cu(I)[BCA]2 and the assumed product Cu(II) [measured from copper(II)sulphate in the presence of BCA] were determined under assay conditions to be e482(Cu(I)[BCA]2) = 2882 M1 cm1 and e482(Cu(II)BCA) = 285 M1 cm1 resulting in De482 = 2597 M1 cm1 used for activity determination. All steady state kinetics of ABTS oxidase or laccase activity measurements were fitted with a Michaelis–Menten equation [42]. For the cuprous oxidase data the initial velocity first increases up to a maximum and then falls with increasing substrate concentration. This observation was also described by Djoko and co-workers [23]. They used an extended Michaelis–Menten equation containing a term dependent on ligand (BCA) concentration. The Cu(I)[BCA]2 kinetics data of our work were tried to be fitted by this extended Michaelis–Menten equation proposed by Djoko et al. Although the fitting of our data gave results, the errors indicated an over-interpretation of our data. Therefore, the simpler model of substrate excess inhibition, first described by Murray in 1930 [43], was chosen with v = vmax/(1 + Km/[S]+[S]/Ki). This simplification was applicable since the free ligand BCA concentration depended on the copper(I) concentration applied due to the constant molar ratio of Cu(I):BCA = 1:2.5 used in our assay. As the assay was never driven for more than 10% of starting concentration, the small amount of BCA, which is released upon copper binding to the protein, was negligible. All fits in this work were performed using the Levenberg–Marquardt algorithm [44,45] implemented in Origin 7G software (OriginLab Corporation, Friedrichsdorf, Germany). The catalytic unit (U) is defined as 1 lmol of the test substrate being converted in 1 min under the specified conditions.

Results and discussions Identification and cloning of cueORE The putative gene for the multicopper oxidase CueORE from R. erythropolis was identified in silico by protein BLAST (tBLASTn [46]) using a multicopper oxidase CueOEC gene from E. coli K12 as a query (P36649 in UniProt [47]), and restricting results to sequences originating from R. erythropolis. Two provisional protein

sequences were revealed: One from R. erythropolisSK121 (NCBI gi:229491017) and one from strain PR4 (NCBI gi:226305791). Both were so far only annotated automatically from the respective genome sequences gi:229490947 and gi:226303489. Oligonucleotides based on the published nucleotide sequences were successfully used for amplification and cloning of the putative cueORE gene from R. erythropolisDSM311 into vector pET22b. The amino acid sequence of CueORE DSM311 (available at DSMZ, Braunschweig, Germany) as used in this work is 98% and 99% identical to the annotated sequences of R. erythropolis PR4 and SK121, respectively, including the signal peptide. The novel CueORE (499 aa including tag and spacer) from Gram-positive R. erythropolis displays 35% amino acid identity and 49% similarity to CueOEC (516 aa, 56.5 kDa) from Gram-negative E. coli (Fig. 4, also see Supplemental information). Recombinant expression and purification of apo-CueORE Apo-CueORE was expressed heterologously in the cytoplasm of E. coli BL21(DE3) and purified to 90% purity (as determined by size exclusion chromatography, data shown in the Supplemental information). Up to 60 mg of purified protein were obtained from 1 L culture (TB medium) after one-step IMAC. The molecular weight, as estimated from SDS-PAGE band size, was about 55 kDa and accurately matched the size of 54.7 kDa calculated from protein sequence by ExPASy ProtParam [48]. The hydrodynamic size of apo-CueORE determined by SEC with globular protein standards was 35 kDa and indicated a monodisperse, monomeric form that was well folded and thereby smaller than calculated. The identity of the protein was confirmed via matrix assisted laser desorption/ionisation mass spectrometry (MALDI-ToFMS, position of used gel pieces is marked by squares in Fig. 1 of Supplemental information, MS-Data are shown in the Supplemental information). Reconstitution of holo-CueORE Following the example of CueOEC, in vitro reconstitution of holoCueORE by incubation of isolated apo-CueORE with copper(II) was investigated according to Djoko et al.[23]. Purified apo-CueORE was incubated over night with different molar excesses of copper(II)sulphate in the presence or absence of 1 mM DTT resulting in intense blue solutions after elution from PD10 desalting columns. For all samples, both the copper content and the specific ABTS oxidation activities were determined. The colourless solution of apo-CueORE without copper(II) addition showed virtually no oxidation activity (see Fig. 2B). However, if copper(II) was added it was stably incorporated and could not be removed by desalting as seen by the blue colour of the protein solutions. As shown in Fig. 2A, copper incorporation depended on the concentration, and data points from both series – with and without DTT – could be fitted to sigmoidal curves with the Hill equation. The effect of DTT can be seen in Table 1; while DTT supplementation effectively had no influence on the maximum molar fraction of copper incorporated into protein (about 53% in both series), the excess amount of copper(II) necessary to obtain half-saturation differed significantly. Without DTT, nearly 11 equivalents of copper(II) were necessary to reach half-saturation in contrast to 3 equivalents in presence of 1 mM DTT. The enhancement of copper binding by means of DTT addition might be caused by reduction of copper(II)ions to copper(I). Although, addition of DTT enhanced copper incorporation; no correlation could be found for ABTS oxidation activity apart from apoCueORE showing no measurable activity at all (see Fig. 2B). The specific activity of reconstituted holo-CueORE was very low with

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A c(Cu)/c(protein) after incubation [%]

Table 1 Fitting results of apo-CueORE reconstitution with copper(II) ions (shown in Fig. 2A). Data were fitted on x = xmaxk/[k + r] with the following parameters: x – molar fraction copper/protein after incubation, xmax – maximum molar fraction copper/protein after incubation, k – copper excess during incubation to reach half-saturation, and r – incubation upon copper excess.

c(Cu)/c(protein) during incubation

spec. activity after inkubation [U/mg]

B

without DTT with DTT

Series

xmax = max. mole fraction copper/ protein [%]

k = incubation copper excess for half-saturation

coefficient of determination (R2)

no DTT 1 mM DTT

52.8 ± 1.1 54.7 ± 0.6

10.6 ± 0.6 3.0 ± 0.2

0.99 0.99

Recombinant expression and purification of holo-CueORE/EC without DTT with DTT

c(Cu)/c(protein) during incubation Fig. 2. In vitro reconstitution of holo-CueORE from purified apo-enzyme. (A) Copper content of holo-protein after desalting in relation to molar copper equivalents during incubation with 1 mM (circles) or without DTT (squares). Data from the colorimetric BCS assay were fitted as sigmoid model with the Hill-equation (x = xmaxk/[k + r]). (B) Specific ABTS activity of the samples. Values are means of duplicates and standard deviations thereof.

approximately 0.05 U/mg protein for all copper(II) additions tested and interpretation of data by fitting was not applicable. Therefore, in vivo formation of holo-CueORE during E. coli expression with copper addition to the culture medium was investigated as an alternative.

For cytosolic expression of holo-CueORE in E. coli, the TB culture medium was supplemented with copper(II)sulphate. The influence of 0–50 mM copper(II) on the expression level and activity of CueORE was tested in an expression series: Concentrations from 10 mM to 50 mM resulted in formation of bluish precipitates (data not shown). The results of quantitative copper determinations before and after E. coli pHT:cueORE cultivation are depicted in Fig. 3A. In general, the measured copper concentration in the medium reflected only 5–10% of the added copper(II)sulphate. The curves of detected copper versus applied copper in the medium were almost parallel for the whole concentration series before and after E. coli cultivation. Slightly lower copper concentrations in the medium after cultivation can be explained with copper uptake of growing E. coli cells expressing CueORE. Poisoning of the bacteria was deduced from prolonged doubling times at 5–15 mM copper(II) and complete growth inhibition at 20 mM and above (data not shown).

Fig. 3. Influence of copper(II) supplementation of growth medium. (A) Uptake of supplemented copper by comparison of copper concentration (BCS assay) in TB medium before inoculation with E. coli BL21(DE3) pHT:cueORE (squares) and after overnight expression (25 °C, 0.1 mM IPTG) (circles). (B) Section of 12% SDS–PAGE showing CueOREbands of overnight cultures with different supplemental copper concentrations. Equal volumes of crude extract loaded; staining by colloidal Coomassie G250 [49]. (C) Volume activity (circles) and specific activity (squares) of IMAC purified CueORE for ABTS substrate plotted against copper(II) supplementation. Standard assay with duplicates. Protein concentration for specific activity was estimated by absorbance A280 assuming 1 mg protein/mL per OD280 unit.

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Both, Fig. 3B and C illustrate that copper(II) supplementation reached a critical concentration at about 10 mM regarding CueORE expression and activity. In Fig. 3B, SDS–PAGE analysis of cultivation samples revealed rather identical expression levels of CueORE in the presence of 5 and 10 mM copper(II). A dramatic decrease occurred above 10 mM since with increasing supplementation the pellets predominantly consisted of copper precipitate rather than bacterial cells. Identical effects were observed for the enzyme activity towards ABTS of IMAC purified samples (see Fig. 3C). CueORE from cells grown in medium without additional copper gave a well detectable band in SDS–PAGE, but showed no measurable volume or specific activity (<0.01 U/mg). However, specific and volume activity of CueORE both correlated with increasing copper(II) concentrations in the medium. Consistent with the SDS– PAGE results, volume activity showed a maximum at 10 mM copper(II) and dropped dramatically beyond that concentration. The apparently unrestricted increase of specific activity is most likely a consequence of protein concentrations being close to 0 mg/mL in the samples of 15 and 20 mM copper additive, which is also reflected by the error bars. As a conclusion of the supplementation experiments, an efficient protocol for in vivo formation of holo-CueORE was established by adding 10 mM copper(II)sulphate to the growth medium, yielding about 15 g cells/L harbouring holo-CueORE with high specific activity. Up to 240 mg of purified protein (1 U/mg, ABTS assay) were obtained from 1 L TB culture after a one-step IMAC. The established expression protocol was applicable for cytosolic expression of CueOEC and its mutants, too. Structural and functional analysis The initial BLAST search using CueOEC as a template identified two CueORE sequences from R. erythropolis strains PR4 and SK121 in the NCBI Protein Databank, which both contain a methionine/ serine rich C-terminal tail. This was also found in the CueORE

cloned in this work from R. erythropolis DSM311. A detailed analysis was not possible by means of bioinformatical methods, because this sequence was identified as a sequence with low complexity and thereby filtered out prior to a BLAST (basic local alignment search tool) by the SEG algorithm [50]. Therefore, a first hint on the possible function came solely from protein sequence alignment of CueORE (DSM311) and CueOEC (E. coli K12, cf. Fig. 4). The sequence of CueORE DSM311 provides a twin-arginine motif, which is clearly identifiable by SignalP-Server [51] same as in CueOEC. The copper binding sites for T1-3 copper found in CueOEC can be found without exception in CueORE (marked in Fig. 4 with triangle, diamond and square symbols). Three plastocyanine like domains could be identified in CueOEC, while in CueORE the identification of only the plastocyanine like domains 1 and 3 was clearly possible; however, the aligned sequences show a high similarity in the area of the second domain. Between domain 2 and 3 of CueOEC, a methionine rich loop is identifiable, containing parts of the T4 copper binding sites. This loop is missing in CueORE generating a gap of 51 pseudo residues length in CueORE between residue 363 and 364. In contrast to this, CueORE showed a C-terminal elongation of 29 amino acids, which contains a repetitive methionine and serine rich motif: (P-M-[D/Q]-M)2-PM-SSSGSHSGH). To check if there is similarity between the methionine rich tail of CueORE and the methionine rich loop of CueOEC, the tail was aligned separately onto CueOEC. Fig. 4 shows the alignment of CueORE493–503 onto CueOEC, which is positioned directly onto the methionine rich loop of CueOEC, where two residues of the T4 binding site are located. These residues can be found in the methionine rich loop of CueORE. While Montfort et al. observed a labile copper (type 4) in a CueOEC crystal structure [26], Djoko et al. showed that this T4 centre might serve as a binding site for copper(I), which is oxidised to copper(II) [23]. Moreover, Sakurai and co-workers [11,52] assumed that the methionine rich region functions as a covering loop for the copper T1 that is responsible for substrate oxidation;

Fig. 4. Comparison of structural elements of CueORE and CueOEC. Sequence alignment of multicopper oxidase sequences of CueOEC of E. coli K12 (Uniprot: P36649) and CueORE from R. erythropolis DSM311. In addition, the methionine rich tail (yellow)MetRE was aligned separately to the sequence of CueOEC. Catalytic important residues are marked with triangles, squares, circles, and diamonds.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Segment of structural alignment with CueOEC (green) with copper type I (T1), labile copper T4 site and T4 copper binding residues [pdb 1N68 [26]], and a homology model of recombinant CueORE (blue). The homology model was created with Modeller [53] implemented in UCSF Chimera [54]. The structure of CueOEC, where the methionine rich loop was deleted [pdb: 2YXW [52], identified as most similar protein in PDB database by BLAST] served as template for modelling. The methionine rich tail region was generated in silico de novo and rotamers were adjusted according to the rotamer database of Dunbrack[55]. This structure was not energy minimized.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Overview of variants of CueORE and CueOEC used in this work. CueO is the multicopper oxidase either from R. erythropolis or E. coli as indicated by the species initials in the index. The suffixes MetEC or MetRE refer to the methionine rich loop of CueOEC or the methionine rich tail of CueORE. A D indicates a deletion of the named part, whereas a single colon indicates a C-terminal fusion and a double colon represents an insertion of a fragment.

removal of this region enhances the laccase activity due to better access of organic substrates. Decreased copper oxidase catalytic efficiency and increased catalytic efficiency for the laccase reaction go along with a drastic reduction of velocity, because of direct T1 exposure to the solvent that results in electron leakage upon deletion of the methionine rich loop. The similarity of the methionine rich region sequences of both species’ CueOs, led us to the hypothesis that the methionine rich tail region of CueORE fulfils the same function as the methionine rich loop from CueOEC. For visualisation, Fig. 5 shows a segment of a homology model of CueORE compared to the structure of CueOEC. The model is based on the loop deletion mutant of CueOEC(pdb: 2YXW [52]) as template, because this was the most similar sequence to CueORE in the protein data bank. For CueORE the methionine rich tail was generated de novo in silico and then connected after amino acid A479. The model accentuated the presence of the putative labile copper T4 binding site formed by D392, M480 and D487 in CueORE. However, the equivalent of CueOEC M441 was missing; at this position a tryptophan occurred in CueORE. Assuming the same spatial position of the methionine rich regions, the additional amino acids between the amino acid that is homologous to CueOEC’s C-terminus (V471) and the putative T4 copper binding site were long enough to connect these two regions. This is achieved by forming a turn starting with L472 ending with P474. The connecting link (G475-P480) pointed nearly

straight towards the methionine rich tail. In this model we did not assume whether this linker has a random coil or b-strand structure. Nevertheless, it is possible to form a methionine rich loop, which might encounter the same function as in E. coli’s cuprous oxidase CueO. Such a tail can be found not just in R. erythropolis. It can also be modelled in silico in various actinobacterial genera such as Rhodococcus, Gordonia, Arthrobacter, Nocardia etc. Based on these data it cannot be distinguished whether this methionine rich tail is a homologue (with position shift in domain architecture) or an analogue to the methionine rich loop of CueOEC. To validate the hypothesis, that the methionine rich tail in CueORE has the same function as the methionine rich loop in CueOEC, we created deletion/insertion chimeras of CueORE and CueOEC and tested them regarding their kinetic properties (cf. Fig. 6). Spectroscopic properties Solutions of all holo-CueORE/EC used in this work showed an intense visible blue colour, whereas the apo-form appeared colourless. Spectra were taken of holo-CueORE/EC and its variants at 25 °C in 20 mM KPi buffer pH 8. All spectra were normalised to the maximum absorption between 599–612 nm (called kblue; Fig. 7A and B). For further analysis of the UV-part have a look in the Supplemental information Table 2.

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A

B

C

Fig. 7. Electronic spectra and CD-spectra. All spectra were taken at 25 °C in 20 mMKPi buffer pH 8. (A) Absorption UV/Vis spectra. (B) Detailed view (315–800 nm) of (A). (C) Mean residue weight (MRW) circular dichroism (CD) spectra after smoothing with Golay–Savitzky algorithm [56]holo-CueORE (solid line), holo-CueOREDMetRE (dotted line) and apo-CueORE (dashed line). Table 2 Spectroscopic characterisation of CuoORE/EC variants. The spectra were normalised to the maximal absorption between 599 and 612 nm (kblue). A peak at 290 nm is covered by the protein peak at 280 nm, an analysis of this peak was carried out by using the slope at this position (derel,290nm/dk). Parameter

CueORE

CueORE DMetRE

CueOEC

CueOEC DMetEC

CueOEC: MetRE

CueOEC DMetEC :MetRE

erel,280nm derel,290nm/dk

14.06 0.15

16.90 0.30

34.15 1.57

12.38 0.31

37.79 1.50

12.92 0.34

kblue [nm];

0.85 599; 1

1.11 597; 1

1.38 610; 1

0.94 612; 1

1.48 611; 1

1.15 609; 1

erel,kblue erel,730nm

0.49

0.5

0.38

0.39

0.43

0.39

-1

[nm ]

erel,320nm

The T3 copper(II) ion was represented by a peak at 320 nm caused by the charge transfer complex OH?Cu(II) with similar intensities around 1 relative unit. All variants of CueORE and CueOEC showed an absorption maximum at around 600 nm caused by the charge transfer complex Sp(Cys)?Cu(II) (T1). The maxima of CueORE variants were at 598 nm, while all CueOEC variants had their maxima at 610 nm indicating that this dative bond is stronger in CueORE [21,28]. Additionally, a brought absorption band at 730 nm was detectable for all variants, while the CueORE mutants had a more intense absorption (0.5 relative units) compared to the 0.4 units caused by the CueOEC variants . Circular dichroism spectroscopy measurements of CueORE variants (Fig. 7C) revealed a high content of b-sheets/turns as well as

T. Classen et al. / Protein Expression and Purification 89 (2013) 97–108

unordered regions in the overall secondary structure composition: 7% a-helix, 38–39% b-sheet for both holo-forms (only 36% for apo-form), 22–23% turns, 31–32% unordered [analysed with CDSSTR algorithm implemented in Dichroweb server [57]. These results compare closely to known atomic structures such as CueO from E. coli [28]. However, the structure of apo-CueORE differed slightly from that of both holo-forms; Holo-CueORE and holoCueOREDMetRE showed a positive Cotton-effect between 195 and 200 nm with a maximal mean residue weight ellipticity (HMRW) of 3000 deg cm2 dmol1. In contrast, the maximum of the apoCueORE spectrum was hypsochromically shifted to 194 nm with HMRW = 1500 deg cm2 dmol1. The zero crossing was at 204 nm for both holo-forms and198 nm for the apo-form. The negative Cotton-effect minimum was located for both holo-proteins at 217 nm (3000 deg cm2 dmol1) while apo-CueORE had its minimum at 212.5 nm (4400 deg cm2 dmol1). This indicates that the overall holo-structure is almost not changed upon the deletion of the first 21 C-terminal amino acids. However, the differences between the apo- and the holo-forms point out that the absence or presence of copper ions bound to the protein had an impact on the overall structure. Activity and stability of holo-CueORE The influence of purification buffer was checked by purifying holo-CueORE in 100 mM MOPS/NaOH, 100 mM TRIS/HCl and 100 mM KPi, respectively, all at pH 8. The specific activities amounted to 0.62 ± 0.08 U/mg for TRIS/HCl, 0.63 ± 0.02 U/mg for MOPS/NaOH, and 0.44 ± 0.03 U/mg for KPi. The protein purified in KPi buffer had just 70% of the activity compared to MOPS or TRIS, while the protein had less blue appearance. Normalisation of these activities with the quotient of A600 nm/A280 nm (U/mg) resulted in nearly identical values: 9.56 ± 1.31 U/mg for TRIS/HCl, 10.43 ± 0.37 U/mg for MOPS/NaOH, and 9.75 ± 0.69 U/mg for

Fig. 8. Effects of temperature and pH on activity and stability of holo-CueORE. For all tests the ABTS oxidation assay was used. Both left (top/bottom) ordinates indicate the level of specific activity at given condition (abscissa). The corresponding values (squares on solid line) are averages from replication with errors bars quantifying the standard deviation. The right ordinates correspond to the half-life of holoCueORE under certain conditions. Values of half-life (circles on dashed lines) are from least-square fits of residual activity in dependence to incubation time, while error bars indicate the 67% uncertainty interval for this value. Top diaGram: Temperature dependence on activity and stability at pH 8. Bottom diagram: Dependence of pH on activity and stability at 25 °C. Note that the right ordinate (half-life) is in log10-scale.

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KPi. Since the 600 nm band in the UV-Vis-spectrum of the protein is caused by the T1 copper centre, the analysis clearly demonstrates that the T1 centre was abstracted/precipitated by KPi buffer leading to a diminished activity. (cf. ref. [23]) To investigate the stability and activity with respect to temperature and pH, the ABTS oxidation assay was chosen. Because of the strong absorption coefficient of the ABTS radical, even low activities can be detected properly. Influences of temperature and pH on stability and activity of holo-CueORE are depicted in Fig. 8. Fig. 8 A shows that activity of holo-CueORE increased with temperatures up to 70 °C. At medium temperatures of up to 45 °C the half-life of the protein remained constant at about 2000 min. At higher temperatures the activity dropped rapidly to virtually zero per minute at 70 °C resembling a sigmoid curve with its inflection point at 55 °C. Hence, holoCueORE appeared to be slightly thermo stable with an optimal working range of 25–45 °C. Regarding the influence of pH (Fig. 8), the activity dropped with increasing pH in solution. If pH is expressed as molarity of protons, the activity increased nearly linearly with proton concentration (data not shown). This can be explained by protons required upon reduction of molecular and atomic oxygen, respectively, to form water. However, the stability at low pH values was poor and the half-life increased roughly bi-exponential with increasing pH. The half-life values determined up to pH10 were reliable, while the errors beyond this value were high due to poor fit results caused by virtually no decrease in activity after three weeks. In conclusion, holo-CueORE is strongly alkali stable, but with respect to activity, an acidic working pH is advised.

Kinetic characterization To analyse whether the enzyme primarily functions as a laccase or a cuprous oxidase, steady state kinetics were carried out. As model substrates for the laccase reaction 2,6-DMP and for the cuprous oxidase reaction Cu(I)[BCA]2were chosen. The results of the steady state kinetics are presented in Table 3 and Fig. 9. The kinetic data gained with ABTS and 2,6-DMP were fitted with the Michaelis–Menten equation. The catalytic efficiencies of the CueORE proteins with and without the methionine rich tail (MetRE) were about one order of magnitude higher for the ABTS substrate than those for 2,6-DMP as substrate. The catalytic efficiencies of the CueOEC and CueOEC:MetRE proteins for ABTS were nearly the same (cf. Table 3). However, the catalytic efficiencies for the laccase reaction were twice the ABTS oxidation reaction level. For both CueOECs (CueOECDMetEC, CueOECDMetEC:MetRE) the ABTS conversion efficiency was increased by one order of magnitude upon deletion of the methionine rich loop from E. coli, while the laccase reaction efficiency dropped to the level of 10% compared to those CueOECs with MetEC. It was of no consequence for the catalytic efficiency, if the MetRE was fused to the protein or not. In the cuprous oxidase assays (Table 3), the interpretation of the data with copper(I)[BCA]2as substrate was not possible via the standard Michaelis–Menten equation. Using the Murray fit for substrate excess inhibition [v = vmax/(1 + Km/[S]+[S]/Ki),[43]], very low Km values in the micro-molar range were calculated for both, holoCueORE and holo-CueOREDMetRE, (Table 3). In contrast, the inhibition-constant Ki was four to five times higher than the Km values. Furthermore, the turn-over numbers (kcat) were just moderate with 9.3 min1 for the full-length protein and 2.3 min1 for the truncated version. The catalytic efficiencies (kcat/Km) were very high with >100 min1 mM1. These values were one to two orders higher in magnitude than the catalytic efficiencies for ABTS and 2,6-DMP for both full-length and truncated holo-CueORE. The results serve as indication that the protein is a cuprous oxidase

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Table 3 Results of steady state kinetics of CueORE/EC with substrates ABTS, 2,6-dimethoxyphenol (2,6-DMP), and copper(I)[BCA]2. Kinetic data with ABTS and 2,6-DMP were fitted with the least square algorithm on the Michaelis–Menten equation v = vmax[S]/(Km[S]) with v – resulting initial velocity, vmax – maximal velocity, [S] – initial substrate concentration, and Km – Michaelis-constant. Data gained with copper(I)[BCA]2 were fitted with an equation describing substrate excess inhibition [43] v = vmax/(1 + Km/[S]+[S]/Ki) with the additional parameter Ki being the inhibition constant. Error bars given are 67% interval estimations of uncertainty obtained from fitting algorithm and propagated errors in case of kcat/Km values. Substrate

Parameter

CueORE

CueORE DMetRE

CueOEC

CueOEC:MetRE

CueOEC DMetEC:MetRE

CueOEC DMetEC

ABTS

kcat [min1] Km [mM] kcat/Km [min1mM1] R2 kcat [min1] Km [mM] kcat/Km [min1 mM1] R2 kcat [min1] Km [mM] Ki [mM] kcat/Km [min1mM1] R2

1329.2 ± 81.8 218.7 ± 18.1 6.1 ± 0.9 0.99 0.15 ± 0.02 0.34 ± 0.09 0.44 ± 0.16 0.92 9.3 ± 0.9 0.08 ± 0.013 0.039 ± 0.006 114.9 ± 29.8 0.79

563.0 ± 68.2 73.9 ± 16.5 7.6 ± 2.6 0.98 0.16 ± 0.01 0.24 ± 0.04 0.64 ± 0.13 0.97 2.3 ± 0.7 0.02 ± 0.01 0.08 ± 0.04 118.8 ± 110.82 0.74

107.2 ± 5.1 20.2 ± 2.8 5.3 ± 1.0 0.98 29.85 ± 2.27 2.72 ± 0.98 10.98 ± 4.81 0.91 651a ± 37 0.169a ± 0.024

205.67 ± 15.43 19.7 ± 4.4 10.4 ± 3.1 0.94 86.00 ± 5.88 4.45 ± 1.63 19.34 ± 8.43 0.96 – – – – –

3975.7 ± 151.1 21.1 ± 2.3 188.8 ± 28.1 0.99 4.70 ± 0.89 2.83 ± 0.80 1.66 ± 0.79 0.97 – – – – –

4488.6 ± 124.2 19.0 ± 1.6 236.7 ± 26.4 0.99 0.67 ± 0.04 0.18 ± 0.03 3.78 ± 0.91 0.94 – – – – –

2,6-DMP

Cu(I) [BCA]2

3852a ± 765.9 –

a Values taken from ref. [27]; pH 5 instead of 5.5 and determined using an oxygen electrode instead of a colorimetric assay; thereby a Michaelis–Menten-fit without inhibition was carried out.

Fig. 9. Catalytic efficiencies (kcat/Km)of CueORE/EC variants for ABTS and 2,6dimethoxyphenol, all taken at 30 °C in duplicates. Grey bars – ABTS kinetics assay conditions: 5–99 mM ABTS in 100 mM sodium acetate buffer pH5.5, k = 420 nm, e420 = 36,000 M1 cm1[28]. White bars – Laccase kinetics assay conditions: 0.025– 100 mM 2,6-Dimetoxyphenol in 50 mM sodium acetate buffer pH 5.5, 50% (v/v) acetonitrile, k = 477 nm, e477 = 14,800 M1 cm1[28].

rather than a laccase, even though the protein is capable to perform laccase reaction.

have facilitated the incorporation of the metal ions. This can occur by reducing cystine to cysteines thereby providing access of copper to the binding site. However, the option is not possible because there is just one cysteine present in the CueORE primary structure. Alternatively, it can be explained by the reduction of copper(II) cations to copper(I), which must be incorporated to form the fully functional oxido-reductase. Nevertheless, in vitro copper incorporation is not trivial and needs further elucidation. In the ABTS oxidation assay, the in vivo generated holo-CueORE had an activity of approximately 1 U/mg and was significantly more active than the in vitro formed, reconstituted holo-CueORE (0.05 U/mg).Therefore, a new protocol for direct heterologous expression of holo-CueORE in E. coli was established: The in vivo formation of the protein was triggered by the addition of copper(II) and a supplementation of 10 mM copper(II)proved to be optimal (Fig. 2B). The in vivo protocol was also feasible for the expression of holo-CueOEC. Here, we think this could be a general protocol for holo-formation of bacterial multicopper oxidase with high activities.

Activity and stability of holo-CueORE The cytosolically expressed holo-CueORE showed slight thermo stability up to 55 °C and an extreme alkaline stability at pH 12. The enzyme provided a high activity of up to5 U/mg at acidic pH values, whereas the activity was virtually zero above pH 8.

Conclusion

Structure and the role of the methionine tail

Comparison of apo- and holo-CueORE

Using CD-spectroscopy and UV-VIS-spectroscopy we could clearly show structural and electronic similarities between CueORE and its known homologue from E. coli. However, electronical spectra from CueORE differed in the position of the ‘‘blue’’ maximum. With 599 nm this was shifted about 10 nm hypsochromically relative to the maximum in CueOEC. The MetRE did not confer a change in the spectroscopic properties upon deletion/insertion as it was observed with MetEC. This rejected the hypothesis, that MetRE forms a labile T4-binding site like MetEC does. On the other hand, the deletion/insertion variants of MetRE varied significantly the catalytic properties relative to the wildtypes, although the overall catalytic efficiency remained unaffected. In summary, the function of the methionine rich tail remains enigmatic and further studies such as an atomic structure might elucidate its mode of action.

In the present endeavour, we showed the successful heterologous expression of CueORE in both the apo- and the holo-form in E. coli. Apo-CueORE was partially converted to the active holo-form by addition of copper(II) ions. Here, the addition of DTT enhanced the copper binding significantly but could not increase the maximum fraction of active protein (Fig. 2A). However, assuming that at least four copper ions per molecule of CueORE have to be bound to result in an active enzyme (one T1, one T2, two T3 coppers, and optionally a labile T4 copper), a fraction of 0.5 mole copper to protein was quite low, but these results were in accordance to Li et al. [58], where the copper content in CueOEC was determined by X-ray crystallography. The in situ reduction of copper(II) by DTT might

T. Classen et al. / Protein Expression and Purification 89 (2013) 97–108

Acknowledgments We gratefully acknowledge the Ministry of Innovation, Science and Research of the German federal state of North Rhine-Westphalia (technology platform ‘ExpressO’ within the ‘Ziel 2-ProGramm 2007-2013, NRW – EFRE’, and NRW Research School BioStruct) and the‘Gründerstiftung zur Förderung von Forschung und wissenschaftlichemNachwuchsan der Heinrich-Heine-Universität Düsseldorf (NRW Research School BioStruct). We thank Andreas Küberl for his technical support (mass spectrometry of the proteins), Vlada Urlacher, Elisa Classen, Sonja Meyer zu Berstenhorst, and Panida Pollawit for reviewing the manuscript, and Georg Groth as well as Holger Gohlke for very helpful discussions. Furthermore, we acknowledge the entire technical staff of IBOC for their ongoing support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2013.02.003. References [1] M.F. Hullo, I. Moszer, A. Danchin, I. Martin-Verstraete, CotA of Bacillus subtilis is a copper-dependent laccase, J. Bacteriol. 183 (2001) 5426–5430. [2] G. Benfield, S.M. Bocks, K. Bromley, B. Brown, Studies of fungal and plant laccases, Phytochemistry 3 (1964) 79–88. [3] S. Kumar, P.S. Phale, S. Durani, P.P. Wangikar, Combined sequence and structure analysis of the fungal laccase family, Biotechnol. Bioeng. 83 (2003) 386–394. [4] B. Valderrama, P. Oliver, A. Medrano-Soto, R. Vazquez-Duhalt, Evolutionary and structural diversity of fungal laccases, Antonie Van Leeuwenhoek 84 (2003) 289–299. [5] P. Baldrian, Fungal laccases – occurrence and properties, FEMS Microbiol. Rev. 30 (2006) 215–242. [6] P.J. Hoegger, S. Kilaru, T.Y. James, J.R. Thacker, U. Kues, Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences, FEBS J. 273 (2006) 2308–2326. [7] W.G. Zumft, D.J. Gotzmann, P.M. Kroneck, Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens, Eur. J. Biochem. 168 (1987) 301–307. [8] J. Ohkawa, N. Okada, A. Shinmyo, M. Takano, Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression, Proc. Natl. Acad. Sci. USA 86 (1989) 1239–1243. [9] F.F. Fenderson, S. Kumar, E.T. Adman, M.Y. Liu, W.J. Payne, J. LeGall, Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes, Biochemistry 30 (1991) 7180–7185. [10] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Multicopper oxidases and oxygenases, Chem. Rev. 96 (1996) 2563–2606. [11] S. Kurose, K. Kataoka, K. Otsuka, Y. Tsujino, T. Sakurai, Promotion of laccase activities of Escherichia coli cuprous oxidase, CueO by deleting the segment covering the substrate binding site, Chem. Lett. 36 (2007) 232–233. [12] S. Riva, Laccases: blue enzymes for green chemistry, Trends Biotechnol. 24 (2006) 219–226. [13] S. Witayakran, A.J. Ragauskas, Synthetic applications of laccase in green chemistry, Adv. Synth. Catal. 351 (2009) 1187–1209. [14] H. Leutbecher, S. Hajdok, C. Braunberger, M. Neumann, S. Mika, J. Conrad, U. Beifuss, Combined action of enzymes: the first domino reaction catalyzed by Agaricus bisporus, Green Chem. 11 (2009) 676–679. [15] K. Koschorreck, S.M. Richter, A. Swierczek, U. Beifuss, R.D. Schmid, V.B. Urlacher, Comparative characterization of four laccases from Trametes versicolor concerning phenolic C–C coupling and oxidation of PAHs, Arch. Biochem. Biophys. 474 (2008) 213–219. [16] J. Pietruszka, C. Wang, Laccase-catalyzed 3-Arylation of 3-Substituted Oxindoles, ChemCatChem 4 (2012) 782–785. [17] H. Claus, Laccases and their occurrence in prokaryotes, Arch. Microbiol. 179 (2003) 145–150. [18] P. Sharma, R. Goel, N. Capalash, Bacterial laccases, World J. Microbiol. Biotechnol. 23 (2007) 823–832. [19] K. Koschorreck, S.M. Richter, A.B. Ene, E. Roduner, R.D. Schmid, V.B. Urlacher, Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids, Appl. Microbiol. Biotech. 79 (2008) 217–224. [20] E. Rodriguez, M.A. Pickard, R. Vazquez-Duhalt, Industrial dye decolorization by laccases from ligninolytic fungi, Curr. Microbiol. 38 (1999) 27–32. [21] T. Sakurai, K. Kataoka, Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase, Chem. Rec. 7 (2007) 220–229.

107

[22] L. Ausec, J.D. van Elsas, I. Mandic-Mulec, Two-and three-domain bacterial laccase-like genes are present in drained peat soils, Soil Biol. Biochem. 43 (2011) 975–983. [23] K.Y. Djoko, L.X. Chong, A.G. Wedd, Z. Xiao, Reaction mechanisms of the multicopper oxidase CueO from Escherichia coli support its functional role as a cuprous oxidase, J. Am. Chem. Soc. 132 (2010) 2005–2015. [24] G. Grass, C. Rensing, CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli, Biochem. Biophys. Res. Commun. 286 (2001) 902– 908. [25] C. Rensing, G. Grass, Escherichia coli mechanisms of copper homeostasis in a changing environment, FEMS Microbiol. Rev. 27 (2003) 197–213. [26] S.A. Roberts, G.F. Wildner, G. Grass, A. Weichsel, A. Ambrus, C. Rensing, W.R. Montfort, A labile regulatory copper ion lies near the T1 copper site in the multicopper oxidase CueO, J. Biol. Chem. 278 (2003) 31958–31963. [27] S.K. Singh, G. Grass, C. Rensing, W.R. Montfort, Cuprous oxidase activity of CueO from Escherichia coli, J. Bacteriol. 186 (2004) 7815–7817. [28] K. Kataoka, H. Komori, Y. Ueki, Y. Konno, Y. Kamitaka, S. Kurose, S. Tsujimura, Y. Higuchi, K. Kano, D. Seo, T. Sakurai, Structure and function of the engineered multicopper oxidase CueO from Escherichia coli-deletion of the methioninerich helical region covering the substrate-binding site, J. Mol. Biol. 373 (2007) 141–152. [29] A.K. Wernimont, D.L. Huffman, L.A. Finney, B. Demeler, T.V. O’Halloran, A.C. Rosenzweig, Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli, J. Biol. Inorg. Chem. 8 (2003) 185–194. [30] S. Silver, L.T. Phung, Bacterial heavy metal resistance: new surprises, Annu. Rev. in Microbiol. 50 (1996) 753–789. [31] W. Donovan, L. Zheng, K. Sandman, R. Losick, Genes encoding spore coat polypeptides from Bacillus subtilis, J. Mol. Biol. 196 (1987) 1–10. [32] M. Letek, P. González, I. MacArthur, H. Rodríguez, T.C. Freeman, A. Valero-Rello, M. Blanco, T. Buckley, I. Cherevach, R. Fahey, The genome of a pathogenic Rhodococcus: cooptive virulence underpinned by key gene acquisitions, PLoS Genet. 6 (2010) e1001145. [33] M.J. Larkin, L.A. Kulakov, C.C.R. Allen, Biodegradation and Rhodococcus – masters of catabolic versatility, Curr. Opin. Biotechnol. 16 (2005) 282–290. [34] I. Kullartz, J. Pietruszka, Cloning and characterisation of a new 2-deoxy-dribose-5-phosphate aldolase from Rhodococcus erythropolis, J. Biotech. (in press). [35] P. Gray, H. Thornton, Soil bacteria that decompose certain aromatic compounds, Zentr Bakt Parasitenk Abt II (78) (1928) 74–96. [36] M. Goodfellow, G. Alderson, J. Lacey, Numerical taxonomy of Actinomadura and related actinomycetes, J. Gen. Microbiol. 112 (1979) 95–111. [37] J. Chiu, P.E. March, R. Lee, D. Tillett, Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h, Nucleic Acids Res. 32 (2004) e174. [38] K. Miyazaki, Creating random mutagenesis libraries by megaprimer PCR of whole plasmid (MEGAWHOP), Methods Mol. Biol. 231 (2003) 23–28. [39] S.D. Moore, P.E. Prevelige Jr., A P22 scaffold protein mutation increases the robustness of head assembly in the presence of excess portal protein, J. Virol. 76 (2002) 10245–10255. [40] C.C. Douglas, D. Thomas, J. Lanman, P.E. Prevelige Jr., Investigation of Nterminal domain charged residues on the assembly and stability of HIV-1 CA, Biochemistry 43 (2004) 10435–10441. [41] C. Follo, C. Isidoro, A fast and simple method for simultaneous mixed sitespecific mutagenesis of a wide coding sequence, Biotechnol. Appl. Biochem. 49 (2008) 175–183. [42] L. Michaelis, M.L. Menten, Die Kinetik der Inwertinwirkung, Biochemistry (1913) 333–369. [43] D.R.P. Murray, The inhibition of esterases by excess substrate, Biochem. J. 24 (1930) 1890–1896. [44] K. Levenberg, A method for the solution of certain problems in least squares, Q. Appl. Math. 2 (1944) 164–168. [45] D.W. Marquardt, An algorithm for least-squares estimation of nonlinear parameters, SIAM J. Appl. Math. 11 (1963) 431–441. [46] S.F. Altschul, T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [47] E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel, A. Bairoch, Protein identification and analysis tools on the ExPASy server, Springer, 2005. [48] E. Jain, A. Bairoch, S. Duvaud, I. Phan, N. Redaschi, B. Suzek, M. Martin, P. McGarvey, E. Gasteiger, Infrastructure for the life sciences: design and implementation of the UniProt website, BMC Bioinformatics 10 (2009) 136. [49] N. Dyballa, S. Metzger, Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels, J. Vis. Exp. (2009) 1431–1434. [50] J.C. Wootton, S. Federhen, Statistics of local complexity in amino acid sequences and sequence databases, Comput. Chem. 17 (1993) 149–163. [51] T.N. Petersen, S. Brunak, G. Heijne, H. Nielsen, SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods 8 (2011) 785–786. [52] K. Kataoka, H. Komori, Y. Ueki, Y. Konno, Y. Kamitaka, S. Kurose, S. Tsujimura, Y. Higuchi, K. Kano, D. Seo, Structure and function of the engineered multicopper oxidase CueO from Escherichia coli – deletion of the methionine-rich helical region covering the substrate-binding site, J. Mol. Biol. 373 (2007) 141–152. [53] A. Sali, T. Blundell, Comparative protein modelling by satisfaction of spatial restraints, IOS Press, Amsterdam, The Netherlands, 1994.

108

T. Classen et al. / Protein Expression and Purification 89 (2013) 97–108

[54] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera – a visualization system for exploratory research and analysis, J. Comput. Chem. 25 (2004) 1605–1612. [55] R.L. Dunbrack, Rotamer libraries in the 21st century, Curr. Opin. Struct. Biol. 12 (2002) 431–440. [56] A. Savitzky, M.J.E. Golay, Smoothing and differentiation of data by simplified least squares procedures, Anal. Chem. 36 (1964) 1627–1639.

[57] A. Lobley, L. Whitmore, B. Wallace, DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra, Bioinformatics 18 (2002) 211–212. [58] X. Li, Z. Wei, M. Zhang, X. Peng, G. Yu, M. Teng, W. Gong, Crystal structures of E. coli laccase CueO at different copper concentrations, Biochem. Biophys. Res. Commun. 354 (2007) 21–26.