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• W2066948618 abstract "The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at ≤pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent Km values for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μm, respectively, with corresponding kcat values of 16.8 ± 0.8 and 3.7 ± 0.1 s−1. Maximal enzyme activity was observed at 75 °C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 °C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable. The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at ≤pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent Km values for ABTS and SGZ at 37 °C were of 106 ± 11 and 26 ± 2 μm, respectively, with corresponding kcat values of 16.8 ± 0.8 and 3.7 ± 0.1 s−1. Maximal enzyme activity was observed at 75 °C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 °C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable. Bacterial endospores are cellular structures designed to resist to a wide range of physical-chemical extremes such as wet and dry heat, desiccation, radiation, UV light, and oxidizing agents, would promptly destroy vegetative cells. The remarkable level of resistance of the bacterial endospore is largely attributed to its unique structural features (1Driks A. Microbiol. Mol. Biol. Rev. 1999; 63: 1-20Crossref PubMed Google Scholar, 2Henriques A.O. Moran C.P., Jr. Methods. 2000; 20: 95-110Crossref PubMed Scopus (176) Google Scholar). In Bacillus subtilis the dehydrated spore core, which contains a copy of the chromosome, is surrounded by a thick layer of a modified peptidoglycan called the cortex, which is essential for heat resistance. The cortex is protected from the action of lysozyme and harsh chemicals by a multi-layered protein coat, which also influences the spore response to germinants (1Driks A. Microbiol. Mol. Biol. Rev. 1999; 63: 1-20Crossref PubMed Google Scholar, 2Henriques A.O. Moran C.P., Jr. Methods. 2000; 20: 95-110Crossref PubMed Scopus (176) Google Scholar). In B. subtilis the coat is structurally differentiated into a thin lamellar inner layer closely apposed to the cortex peptidoglycan and a thicker, striated and electron-dense outer layer (1Driks A. Microbiol. Mol. Biol. Rev. 1999; 63: 1-20Crossref PubMed Google Scholar, 2Henriques A.O. Moran C.P., Jr. Methods. 2000; 20: 95-110Crossref PubMed Scopus (176) Google Scholar). The structure of the coat results from a multistep assembly process that involves the temporally and spatially regulated synthesis of more than 30 different protein components ranging in size from about 6 to 70 kDa (1Driks A. Microbiol. Mol. Biol. Rev. 1999; 63: 1-20Crossref PubMed Google Scholar, 2Henriques A.O. Moran C.P., Jr. Methods. 2000; 20: 95-110Crossref PubMed Scopus (176) Google Scholar, 3Stragier P. Losick R. Annu. Rev. Genet. 1996; 30: 297-341Crossref PubMed Scopus (510) Google Scholar). Their order of assembly and final destination within the coat layers appears to rely on a complex pattern of specific protein-protein interactions as well as on a variety of post-translational modifications, including proteolytic processing, cross-linking, and glycosylation (1Driks A. Microbiol. Mol. Biol. Rev. 1999; 63: 1-20Crossref PubMed Google Scholar, 2Henriques A.O. Moran C.P., Jr. Methods. 2000; 20: 95-110Crossref PubMed Scopus (176) Google Scholar). Several components of the B. subtilis spore coat are enzymes, with possible roles in the post-translational modifications that accompany the macromolecular assembly of the coat or in the final resistance properties of the spore structure. For example, a manganese-dependent catalase is a component of the inner coat layers (4Henriques A.O. Beall B.W. Roland K. Moran C.P., Jr. J. Bacteriol. 1995; 177: 3394-3406Crossref PubMed Google Scholar, 5Seyler R. Henriques A.O. Ozin A. Moran C.P., Jr. Mol. Microbiol. 1997; 25: 955-966Crossref PubMed Scopus (47) Google Scholar), and a transglutaminase associates with the outer coat layers to promote ε-(γ-Glu)Lys cross-linking of specific structural components (6Kobayashi K. Kumazawa Y. Miwa K. Yamanaka S. FEMS Microbiol. Lett. 1996; 144: 157-160Google Scholar, 7Suzuki S. Izawa Y. Kobayashi K. Eto Y. Yamanaka S. Kubota K. Yokozeki K. Biosci. Biotechnol. Biochem. 2000; 64: 2344-2351Crossref PubMed Scopus (54) Google Scholar). Another enzyme that appears to associate with the outer coat layers is the 65-kDa product of the cotA gene (8Donovan W. Zheng L. Sandman K. Losick R. J. Mol. Biol. 1987; 196: 1-10Crossref PubMed Scopus (136) Google Scholar, 9Zheng L. Donovan W.P. Fitz-James P.C. Losick R. Genes Dev. 1988; 2: 1047-1054Crossref PubMed Scopus (167) Google Scholar, 10Zheng L. Losick R. J. Mol. Biol. 1990; 212: 645-660Crossref PubMed Scopus (118) Google Scholar). CotA belongs to a diverse group of multi-copper “blue” oxidases that includes the laccases (11Alexandre G. Zhulin I.B. Trends Biotechnol. 2000; 18: 41-42Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Purified wild type B. subtilis spores but not those of a cotA insertional mutant are able to oxidize the laccase substrates SGZ 1The abbreviations used are: SGZsyringaldazineABTS2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)T1type 1 copperT2type 2 copperT3type 3 copperZAOzucchini ascorbate oxidaseLBLuria-BertaniDSMDifco sporulation mediumIPTGisopropyl-β-d-thiogalactopyranosideEPRelectron paramagnetic resonancebpbase pair(s) 1The abbreviations used are: SGZsyringaldazineABTS2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)T1type 1 copperT2type 2 copperT3type 3 copperZAOzucchini ascorbate oxidaseLBLuria-BertaniDSMDifco sporulation mediumIPTGisopropyl-β-d-thiogalactopyranosideEPRelectron paramagnetic resonancebpbase pair(s) and ABTS (12Henriques A.O. Martins L.O. Soares C.M. Costa T. Pereira M.M. Teixeira M. J. Biol. Inorg. Chem. 2001; 86: 259Google Scholar,13Hullo M.-F. Moszer I. Danchin A. Martin-Verstraete I. J. Bacteriol. 2001; 183: 5426-5430Crossref PubMed Scopus (318) Google Scholar). The absence of CotA has no detectable effect on lysozyme resistance or germination, but it does prevent the appearance of a brown pigment characteristic of colonies in the late stages of sporulation (8Donovan W. Zheng L. Sandman K. Losick R. J. Mol. Biol. 1987; 196: 1-10Crossref PubMed Scopus (136) Google Scholar, 14Rogolsky M. J. Bacteriol. 1968; 95: 2426-2427Crossref PubMed Google Scholar), which appears to protect spores against UV light (13Hullo M.-F. Moszer I. Danchin A. Martin-Verstraete I. J. Bacteriol. 2001; 183: 5426-5430Crossref PubMed Scopus (318) Google Scholar). Laccases (EC 1.10.3.2) catalyze the oxidation of a variety of aromatic compounds, in particular phenolic substrates, coupled to the reduction of molecular oxygen to water (15Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2605Crossref PubMed Scopus (3105) Google Scholar). Their catalytic centers consist of three structurally and functionally distinct copper centers. T1 copper (“blue copper”), is a mononuclear center involved in substrate oxidation, whereas both T2 and T3 form a trinuclear center involved in the oxygen reduction to water (15Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2605Crossref PubMed Scopus (3105) Google Scholar). Laccases are receiving increased attention as a model system for characterizing the structure-function relationship of copper-containing proteins because of their potential for biotechnological applications in fields such as delignification, plant fiber derivatization, textile dye or stain bleaching, and contaminated water or soil detoxification (16Breen A. Singleton F.L. Curr. Opin. Biotechnol. 1999; 10: 252-258Crossref PubMed Scopus (151) Google Scholar). These enzymes are widely distributed in plants and fungi, where they have been implicated in melanin formation, lignolysis, and detoxification (17Thurston C.F. Microbiology. 1994; 140: 19-26Crossref Scopus (1610) Google Scholar). Several protein sequences with significant similarity to fungal laccases have been predicted in bacterial genomes (11Alexandre G. Zhulin I.B. Trends Biotechnol. 2000; 18: 41-42Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), but other than in B. subtilis spores (12Henriques A.O. Martins L.O. Soares C.M. Costa T. Pereira M.M. Teixeira M. J. Biol. Inorg. Chem. 2001; 86: 259Google Scholar, 13Hullo M.-F. Moszer I. Danchin A. Martin-Verstraete I. J. Bacteriol. 2001; 183: 5426-5430Crossref PubMed Scopus (318) Google Scholar), laccase activity was found in only three other bacterial species, the soil bacterium Azospirillum lipoferum (18Givaudan A. Efosse A. Faure D. Potier P. Bouillant M.-L. Bally R. FEMS Microbiol. Lett. 1993; 108: 205-210Crossref Scopus (24) Google Scholar) and the marine bacteria Marinomonas mediterranea and strain 2-40 (19Sanchez-Amat A. Solano F. Biochem. Biophys. Res. Commun. 1997; 240: 787-792Crossref PubMed Scopus (92) Google Scholar, 20Solano F. Garcı́a E. Pérez de Egea E. Sanchez-Amat A. Appl. Environ. Microbiol. 1997; 63: 3499-3506Crossref PubMed Google Scholar, 21Sanchez-Amat A. Lucas-Elio P. Férnandez E. Garcia-Borrón J.C. Solano F. Biochim. Biophys. Acta. 2001; 1547: 104-116Crossref PubMed Scopus (95) Google Scholar). However, to date no bacterial laccase has been purified and characterized in detail. syringaldazine 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) type 1 copper type 2 copper type 3 copper zucchini ascorbate oxidase Luria-Bertani Difco sporulation medium isopropyl-β-d-thiogalactopyranoside electron paramagnetic resonance base pair(s) syringaldazine 2′2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) type 1 copper type 2 copper type 3 copper zucchini ascorbate oxidase Luria-Bertani Difco sporulation medium isopropyl-β-d-thiogalactopyranoside electron paramagnetic resonance base pair(s) Here, we have shown that CotA has all the molecular features typical of ascorbate oxidase and fungal laccase, namely an exposed reactive center, and confirmed that CotA oxidase activity is directly required for the formation of spore pigment. Furthermore we overproduced, purified, and characterized biochemically the recombinant enzyme and found spectroscopic and kinetic properties consistent with those reported for fungal laccases. We show that both the spore-associated enzyme or the purified protein are remarkably heat stable. CotA is naturally associated with the coat structure in an active form, and expression of cotA from a multicopy plasmid results in spores with greatly increased levels of CotA. Therefore, we suggest that the B. subtilis endospore coat structure can be used as a surface display system for biocatalyst applications involving the highly stable CotA laccase. The structures of ascorbate oxidase from zucchini (Ref. 22Messerschmidt A. Landenstein R. Huber R. Bolognesi M. Avigliano L. Petruzzelli R. Rossi A. Finazzi-Agró A. J. Mol. Biol. 1992; 224: 179-205Crossref PubMed Scopus (438) Google Scholar; PDB code 1AOZ) and laccase from Coprinus cinereus (Ref. 23Ducros V. Brzozowski A.M. Wilson K.S. Brown S.H. Ostergaard P. Schneider P. Yaver D.S. Pedersen A.H. Davies G.J. Nat. Struct. Biol. 1998; 5: 310-316Crossref PubMed Scopus (344) Google Scholar; PDB code 1A65) were used to derive a structural model of CotA by comparative modeling techniques. The program Modeler version 4 (24Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10294) Google Scholar) was used for this purpose. The ZAO and laccase structures were superimposed to generate a sequence alignment that reflected the equivalence of residues in the structure (that may differ from common sequence alignments). The CotA sequence was then aligned against the primary alignment. Because of the low sequence identity, this alignment had a considerable number of ambiguities, which were taken into account in the final model (see “Results”). The initial alignment of CotA was used to generate structural models that were then checked using two criteria, identification of zones displaying restrain violations using Modeler and checking several stereochemical and conformational criteria using PROCHECK (25Laskowski A. MacArthur M. Moss D. Thorton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The alignment was changed to correct these problems, and a new cycle was started. Several of these cycles were performed to optimize the alignment and the structural models obtained. Forty structural models were generated with the final alignment and the model displaying the lowest value of the objective function was chosen as the final structural model for CotA. The same procedure used for the wild type was implemented to model the structures of the proteins bearing point mutations in the T1 center. The optimized alignment of the wild type was changed in the specified position of the mutation, and the model with the lowest value of the objective function was chosen from 40 generated structural models. Two mutants are described in this work, H497A and M502L, both affecting the type I copper site. For modeling the structure of the M502L mutant, the structure of the T1 center of laccase (with its ligands) was used. The B. subtilis strains used in this study are listed in Table I. They are congenic derivatives of the wild type MB24 strain (Table I). The Escherichia coli strain DH5α (laboratory stock) was used for routine cloning procedures, propagation, and amplification of all plasmid constructs. Overproduction of the CotA protein was performed in E. coli strain Tuner (DE3) (Novagen). The E. coli and B. subtilis strains were routinely grown and maintained in LB medium with appropriate antibiotic selection when needed. Sporulation of B. subtilis was induced by growth and exhaustion in Difco sporulation medium (4Henriques A.O. Beall B.W. Roland K. Moran C.P., Jr. J. Bacteriol. 1995; 177: 3394-3406Crossref PubMed Google Scholar). Whenever required, CuCl2 was added to LB or DSM liquid or solid media, as specified in the text.Table IBacterial strains used in this studyStrain or plasmidGenotype or propertiesReference or sourceE. coli Tuner (DE3)PT7lac promoterNovagen AH3520Tuner (DE3) (pET21a(+))1-aReplicative plasmids are shown in parenthesis.This work AH3517Tuner (DE3) (pLOM10)This workB. subtilis MB24trpC2 metC3/Spo+(wild type)Laboratory stock AH76trpC2 metC3 cotA::cm/CmrLaboratory stock AH3512MB24ΩpLOM8′2/SprThis work AH3513MB24ΩpLOM9/SprThis work AH3514MB24ΩpLOM2/SprThis work AH2734MB24 (pTC66)/SprThis work AH2350MB24 (pMK3)/SprRef. 29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholarb The omega symbol (Ω) denotes that the plasmid was integrated into the B. subtilis chromosome by a Campbell integration event (single reciprocal crossover) in the cotA region of homology.1-a Replicative plasmids are shown in parenthesis. Open table in a new tab b The omega symbol (Ω) denotes that the plasmid was integrated into the B. subtilis chromosome by a Campbell integration event (single reciprocal crossover) in the cotA region of homology. The 3′-end of the wild type cotA gene was PCR-amplified from chromosomal DNA of strain MB24 (Table I) using primers cotA-1189D (5′-CAGATGCATATATCATGCAATTCAGAGTC-3′) and cotA-1892R (5′-TCATGTAGATCTTGTGTGAGCATAAAAAGCAGCTCC-3′). The resulting 703-bp DNA fragment was purified, digested with NsiI and BglII, and cloned between the same sites in pMS38 2M. Serrano, R. Fior, C. P. Moran, Jr., and A. O. Henriques, unpublished results. to produce pLOM2. Plasmid pLOM2 served as a template for site-directed mutagenesis. Single amino acid substitutions in copper center I (histidine 479 to an alanine, H479A, or of methionine 502 to a leucine, M502L) were created using the QuikChange system (Stratagene). Primers cotA-HAD (5′-GCCATATTCTAGAGGCGGAAGACTATGACATG-3′) and cotA-HAR (5′-CATGTCATAGTCTTCCGCCTCTAGAATATGGC-3′) were used to create the H479A mutation, whereas primers cotA-MLD (5′-GCATGAAGACTATGACCTGATGAGACCGATGG-3′) and cotA-MLR (5′-CCATCGGTCTCATCAGGTCATAGTCTTCATGC-3′) were used to generate the M502L mutation. The presence of the desired mutations in the resulting plasmids, pLOM8 (carrying the H479A allele) and pLOM9 (bearing the M502L mutation), and the absence of unwanted mutations in other regions of the insert or in pLOM2 (wild type cotA sequence) were confirmed by sequencing. Competent cells of B. subtilis MB24 were transformed with plasmids pLOM8, pLOM9, and pLOM2 with selection for chloramphenicol resistance (Cmr). Transformants were expected to arise as the result of a single reciprocal recombination event (Campbell-type mechanism) between the cloned DNA and the corresponding region of homology in the host chromosome (see Fig. 1). For each cross, transformants were identified with the expected structure in the vicinity of the cotA locus, as determined by PCR with appropriate primers. Most of the crossovers were expected to occur upstream of the mutation, because the insert in the plasmid extends by about 500 bp upstream of this position and only some 200 bp downstream of this position (see Fig. 1). Crossovers upstream of the mutation would result in cells expressing only the mutant cotA allele. To establish that the crossover had generated a full-length mutated copy of the cotA gene, the presence of the correct mutations was confirmed by sequencing appropriate PCR fragments derived from the recombinant chromosomes. In addition, the cotA locus in cells resulting from integration of pLOM2 (which carries the wild type sequence) was also sequenced to confirm that no other mutations had been serendipitously introduced into the chromosome. Strains AH3512, AH3513, and AH3514 were the result of the Campbell integration of pLOM2, pLOM8, and pLOM9 into the cotA locus and express the wild type, cotA H479A, or cotA M502L alleles, respectively (Table I). Primers cotA107D (5′-CGGGCTGCAGCACGAAGATTTTTTG-3′) and cotA-1892R (see above) were used to PCR-amplify a 1785-bp fragment extending 55 bp upstream of the cotA transcription initiation (+1) site (27Sandman K. Kroos L. Cutting S. Youngman P. Losick R. J. Mol. Biol. 1988; 200: 461-473Crossref PubMed Scopus (78) Google Scholar). The resulting PCR product encompasses the entire cotA gene including its promoter flanked by engineered PstI and BglII sites (Fig. 1). The PCR product was digested and cloned between the PstI and BamHI sites of plasmid pMK3 (28Sullivan M.A. Yasbin R.E. Young F. Gene. 1984; 29: 21-26Crossref PubMed Scopus (191) Google Scholar), thereby creating the replicative plasmid pTC66. This plasmid was introduced into the wild type strain MB24 by transformation followed by selection for neomycin resistance. This produced strain AH2734, a congenic derivative of MB24 that bears a multicopy allele of the cotA gene (Table I). Mature spores were harvested 24 h after the onset of sporulation and subjected to a step gradient of Renocal-76 (Bristol-Myers Squibb Co.) for purification (4Henriques A.O. Beall B.W. Roland K. Moran C.P., Jr. J. Bacteriol. 1995; 177: 3394-3406Crossref PubMed Google Scholar). When specified, spores were harvested after treatment of the cultures with lysozyme (12.5 units/ml for 10 min at 37 °C). Occasionally a drop of chloroform was added to facilitate lysis of the mother cell. The analysis of the coat polypeptide composition in wild type and various mutant spores was done as previously described (4Henriques A.O. Beall B.W. Roland K. Moran C.P., Jr. J. Bacteriol. 1995; 177: 3394-3406Crossref PubMed Google Scholar). Briefly, the coat proteins were extracted from 2 A580 units of purified spores by boiling the suspension for 8 min in the presence of 125 mmTris, 4% SDS, 10% (v/v) 2-mercaptoethanol, 1 mmdithiothreitol, 0.05% bromphenol blue, 10% glycerol at pH 6.8 (4Henriques A.O. Beall B.W. Roland K. Moran C.P., Jr. J. Bacteriol. 1995; 177: 3394-3406Crossref PubMed Google Scholar). The extracted proteins were resolved on 15% SDS-PAGE. The gels were then stained with Coomassie Blue, destained, and scanned for analysis. The cotA gene was amplified by PCR using oligonucleotides cotA159D (5′-CTATAGTACTAGTTTGGAAAATTTAG-3′) and cotA-1892R (see above). The 1733-bp-long PCR product was digested with BglII and SpeI and inserted between the BamHI and NheI sites of plasmid pET21a(+) (Novagen) to yield pLOM10. Introduction of pLOM10 into the E. coli strain Tuner (DE3) (Novagen) produced strain AH3517 (Table I) in which the CotA protein could be produced under the control of the T7 lac promoter. Strain AH3517 was grown in LB medium supplemented with 0.25 mm CuCl2 at 30 °C. Growth was followed until the midlog phase (A600 = 0.3), at which time 1 mm IPTG was added to the culture medium. Incubation was continued for further 3–4 h. Cells were harvested by centrifugation (8,000 × g, 15 min, 4 °C). The cell sediment was suspended in Tris-HCl (20 mm, pH 7.6) containing DNase I (10 μg/ml extract), MgCl2 (5 mm), and a mixture of protease inhibitors (CompleteTM, mini EDTA-free protease inhibitor mixture tablets, Roche Molecular Biochemicals). Cells were disrupted in a French pressure cell (at 19,000 p.s.i.) followed by ultracentrifugation (40,000 × g, 1 h, 4 °C) to remove cell debris and membranes. The resulting soluble extract was loaded onto an ion exchange SP-Sepharose column (bed volume 25 ml) equilibrated with Tris-HCl (20 mm, pH 7.6). Elution was carried out with a two-step linear NaCl gradient (0–0.5 and 0.5–1m) in the same buffer. Fractions containing laccase-like activity were pooled, concentrated by ultrafiltration (cutoff of 30 kDa), and equilibrated to 20 mm Tris-HCl (pH 7.6). The resulting sample was applied to a MonoS HR5/5 Column (AmershamBiosciences). Elution was carried out with a two-step linear NaCl gradient (0–0.5 and 0.5–1 m). The active fractions were pooled and desalted. After boiling for 10 min in loading dye (see also “Results”), a single protein band of 65 kDa was revealed by SDS-PAGE (12.5%). All purification steps were carried out at room temperature in a fast protein liquid chromatography system (Åkta fast protein liquid chromatography, Amersham Biosciences). Laccase activity was routinely assayed at 37 °C using the ABTS or SGZ substrates as follows. (a) The assay mixture contained 1 mm ABTS and 100 mm citrate-phosphate buffer (pH 4). Oxidation of ABTS was followed by the absorbance increase at 420 nm (ε = 36,000m −1 cm−1). (b) The assay mixture contained 0.1 mm SGZ (dissolved in ethanol) ABTS and 100 mm citrate-phosphate buffer (pH 6). Oxidation of SGZ was followed by the absorbance increase at 525 nm (ε = 65,000 m −1 cm−1). The copper requirement was tested by adding CuCl2 (0–1 mm) to the standard assay mixtures. Enzyme activity measurements were performed either on a Beckman DU©70 spectrophotometer (Beckman Instruments) or on a Molecular Devices Spectra Max 340 microplate reader with a 96-well plate. All assays were performed in triplicate. Enzyme-specific activity was expressed in nmol or μmol of substrate (ABTS or SGZ) oxidized/min/mg of protein or /A580 of a spore suspension. The protein content was determined by the Bradford assay (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using bovine serum albumin as a standard. The UV-visible absorption spectrum was obtained at room temperature in 20 mm Tris-HCl buffer (pH 7.6) using a Shimadzu UV 3100 spectrophotometer. EPR spectra were measured with a Bruker ESP-380 spectrometer equipped with an Oxford Instruments ESR-900 continuous-flow helium cryostat. EPR spectra obtained under non-saturating conditions were theoretically simulated using the Aasa and Vänngard approach (30Aasa R. Vänngard V.T. J. Magn. Reson. 1975; 19: 308-315Google Scholar). The effect of pH on the activity of the enzyme was determined at 37 °C in 100 mm citrate-phosphate buffer (pH 3.0–7.0) and 100 mm potassium phosphate buffer (pH 7.0–8.0) for the ABTS or SGZ substrates, respectively. The temperature optimum for the activity was determined at temperatures ranging from 22 to 80 °C by measuring ABTS oxidation. Enzyme thermostability was measured at 80 °C by incubating an appropriate amount of purified enzyme (25 μg) in 20 mm Tris-HCl (pH 7.6) or 11 A580units of a spore suspension in water. At appropriate times, samples were withdrawn, cooled, and examined for residual activity using the ABTS oxidation assay at 37 °C (see above). Kinetic parameters for the purified enzyme were determined at 37 °C by using different concentrations of ABTS (10–200 μm) or SGZ (1–100 mm). The reactions were initiated by the addition of 0.1 μg of purified CotA protein, and initial rates were obtained from the linear portion of the progress curve. Kinetic data were determined from Lineweaver-Burk plots assuming that simple Michaelis-Menten kinetics were followed. The N-terminal amino acid sequence of purified recombinant CotA was determined on an Applied Biosystem protein sequencer (Model 477A) at the Instituto de Tecnologia Quı́mica e Biológica microsequencing facility. The copper content of purified recombinant CotA was measured by atomic absorption at the Instituto Superior Técnico (Technical University of Lisbon) chemical analysis facility. The molecular mass of the CotA protein was determined on a gel filtration Superose 6 HR 10/30 column (Amersham Biosciences) equilibrated with 20 mm Tris-HCl buffer (pH 7.6) containing 0.15 m NaCl. Ribonuclease (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), albumin (67 kDa), and aldolase (158 kDa) were used as standards. The isoelectric point was evaluated in a Phast System (Amersham Biosciences) against broad pI standards following the manufacture's instructions. B. subtilis CotA is significantly similar at the primary structure level with multicopper oxidases, a protein family that includes the laccases (11Alexandre G. Zhulin I.B. Trends Biotechnol. 2000; 18: 41-42Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 31Mizuguchi K. Deane C.M. Blundell T.L. Overington J.P. Protein Sci. 1998; 7: 2469-2471Crossref PubMed Scopus (416) Google Scholar). Moreover, CotA shows similarity with the two members of this family whose structure is known, having 19.7% sequence identity and 36.6% similarity with zucchini ascorbate oxidase (22Messerschmidt A. Landenstein R. Huber R. Bolognesi M. Avigliano L. Petruzzelli R. Rossi A. Finazzi-Agró A. J. Mol. Biol. 1992; 224: 179-205Crossref PubMed Scopus (438) Google Scholar) and 22.4% identity and 39.3% similarity with laccase from C. cinereus (23Ducros V. Brzozowski A.M. Wilson K.S. Brown S.H. Ostergaard P. Schneider P. Yaver D.S. Pedersen A.H. Davies G.J. Nat. Struct. Biol. 1998; 5: 310-316Crossref PubMed Scopus (344) Google Scholar). However, based on sequence comparisons, C. cinereus laccase and ZAO are more closely related to each other (30.6% identity and 50% similarity) than to CotA. Nevertheless, the comparison of the amino acid sequences between CotA and these members of the multicopper oxidase family showed that the copper ligands are conserved in CotA. Submission of the CotA sequence to GenTHREADER (32Jones D.T. J. Mol. Biol. 1999; 287: 797-815Crossref PubMed Scopus (782) Google Scholar) revealed, with a confidence level above 99%, both ZAO and laccase as possible folds. Because they rely on various factors beyond sequence similarity, these threading methods reinforced the view that CotA is a member of the multicopper oxidase family. Consequently, the crystal structures of ZAO and laccase from C. cinereus were used to derive a structural model for CotA by comparative modeling techniques. The low overall similarity among CotA, ZAO, and the laccase posed several challenges for deriving its structural model. First," @default.
• W2066948618 created "2016-06-24" @default.
• W2066948618 creator A5001795642 @default.
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• W2066948618 title "Molecular and Biochemical Characterization of a Highly Stable Bacterial Laccase That Occurs as a Structural Component of the Bacillus subtilis Endospore Coat" @default.
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