Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus <i>Phanerochaete chrysosporium</i>

Raíces, Manuel; Paifer, Edenia; Cremata, José A.; Montesino, Raquel; Ståhlberg, Jerry; Divne, Christina; Szabó, I.; Henriksson, Gunnar; Johansson, Gunnar; Pettersson, Göran

doi:10.1016/0014-5793(95)00758-2

Cited by 62 publications

(61 citation statements)

References 32 publications

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“…The prosequence occupies the amino acid channel acting like a plug that prevents substrate binding and perturbs active site geometry. To our knowledge, this is a novel type of proenzyme activation not yet found in proteases (10 -12), flavoproteins (13)(14)(15)(16)(17)(18), and the three-dimensional structures of other zymogens studied so far.…”

Section: Structure Of Paopt-ab Complex and Amino Acid Channel-mentioning

confidence: 83%

“…X-ray crystallographic studies of zymogens and comparisons with their counterparts have identified the structural changes that accompany this conversion. As for flavoproteins, several enzymes are expressed as proforms, such as succinate dehydrogenase from Saccharomyces cerevisiae (13), pea ferredoxin-NADP reductase (14), glucose-fructose oxidoreductase from Zymomonas mobilis (15), and cellobiose dehydrogenase from Phanerochaete chrysosporium (16). The size of the prosequence of these enzymes are 18 residues long to 5-kDa molecular mass.…”

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confidence: 99%

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Structural Basis of Proteolytic Activation of l-Phenylalanine Oxidase from Pseudomonas sp. P-501

Ida

Kurabayashi²,

Suguro

et al. 2008

Journal of Biological Chemistry

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The mature form of L-phenylalanine oxidase (PAOpt) from Pseudomonas sp. P-501 was generated and activated by the proteolytic cleavage of a noncatalytic proenzyme (proPAO). The crystal structures of proPAO, PAOpt, and the PAOpt-o-amino benzoate (AB) complex were determined at 1.7, 1.25, and 1.35 Å resolutions, respectively. The structure of proPAO suggests that the prosequence peptide of proPAO occupies the funnel (pathway) of the substrate amino acid from the outside of the protein to the interior flavin ring, whereas the funnel is closed with the hydrophobic residues at its vestibule in both PAOpt and the PAOpt-AB complex. All three structures have an oxygen channel that is open to the surface of the protein from the flavin ring. These results suggest that structural changes were induced by proteolysis; that is, the proteolysis of proPAO removes the prosequence and closes the funnel to keep the active site hydrophobic but keeps the oxygen channel open. The possibility that an interaction of the hydrophobic side chain of substrate with the residues of the vestibule region may open the funnel as a putative amino acid channel is discussed.L-Phenylalanine oxidase (EC 1.13.12.9: PAO) from Pseudomonas sp. P-501 catalyzes both the oxidative deamination and oxygenative decarboxylation of L-Phe, L-Tyr, L-Trp, and L-Met as a substrate (1-3). The enzyme was screened from bacteria in soil for the clinical determination of L-Phe (4, 5). The enzyme has 2 mol of noncovalent FAD (2) and consists of 2 mol each of ␣ and ␤ subunits (6). Of the above-mentioned amino acids, L-Phe is mainly oxygenated, and L-Met is mainly oxidized by PAO. Kinetic and resonance Raman studies of the enzyme revealed that PAO catalyzes the reaction by the ping-pong type mechanism (7) and that the catalytically important species is the purple intermediate, which consists of a reduced enzyme and an imino acid derived from an amino acid substrate (8). We determined the nucleotide sequence of DNA encoding the enzyme protein (9) (see Fig. 1). The open reading frame encoding a single polypeptide is arranged in the order of initiator Met, a prosequence (14 amino acid residues long), an ␣ subunit (92 amino acid residues long), a dipeptide, and a ␤ subunit (605 amino acid residues long). The polypeptide with the prosequence (proPAO) was successfully expressed in Escherichia coli (9). The purified proPAO contains FAD, but the proenzyme does not show any catalytic activity. Protease (Pronase-trypsin mixture) treatment of proPAO initially removes only the Ile 107 -Lys 108 dipeptide from proPAO, but the removal of this peptide does not lead to the expression of enzyme activity. Further treatment with the protease removes the prosequence from proPAO, and the resultant protein shows activity. The Pronasetrypsin-treated proPAO (PAOpt) 3 showed spectral and kinetic properties comparable with the native enzyme.Several secreted enzymes are expressed as a proform. The prosequence usually acts as an intramolecular chaperone and/or an inhibitor of catalytic activity. The bes...

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Section: Structure Of Paopt-ab Complex and Amino Acid Channel-mentioning

confidence: 83%

mentioning

confidence: 99%

Structural Basis of Proteolytic Activation of l-Phenylalanine Oxidase from Pseudomonas sp. P-501

Ida

Kurabayashi²,

Suguro

et al. 2008

Journal of Biological Chemistry

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“…The deduced amino acid sequence of the GADH dehydrogenase subunit contained three possible glycine boxes, GFGWAG (nt 1036 to 1053), GTGTGG (nt 1282 to 1299), and GAGGAG (nt 2104 to 2121). A homology search of protein databases revealed that the region containing the first glycine box showed sequence similarity with the FAD-binding motif of cellobiose dehydrogenase (CEDH) from Phanerochaete chrysosporium (12,24), sorbose dehydrogenase (SDH) from Gluconobacter oxydans (25), choline dehydrogenase (CDH) of Rhizobium meliloti, glucose dehydrogenase of Drosophila melanogaster (38), human monoamine oxidase B (6), and versicolorin B synthase (VBS) from Aspergillus parasiticus (29). The amino acid sequence alignment is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

1997

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We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay. The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium. Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity. Comparison of the deduced amino acid sequences and the NH 2 -terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively. The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. In E. cypripedii and recombinant E. coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium. The conversion of D-glucose to D-gluconate, 2-keto-D-gluconate (2KDG), and 2,5-diketo-D-gluconate (25DKG) in several species of the genera Acetobacter, Gluconobacter, and Erwinia is mediated by membrane-bound dehydrogenases linked to the cytochrome chains located in the cytoplasmic membrane of the bacterium (1, 31). Membrane-bound gluconate dehydrogenases (GADHs), which catalyze the production of 2KDG from D-gluconate, have been purified and characterized from Pseudomonas aeruginosa (16,19), P. fluorescens (17), Klebsiella pneumoniae (17), Serratia marcescens (17), and Gluconobacter dioxyacetonicus (28). The subunit structures and enzymatic properties of these GADHs were found to be very similar (17). These GADHs consisted of three subunits; namely, flavoprotein, cytochrome c, and a third subunit whose function is unknown. Despite these findings, no further work on gene structures has been reported.In the present work, we describe the direct-expression cloning of the gene cluster encoding three subunits of membranebound GADH from Erwinia cypripedii ATCC 29267. E. coli K-12 derivatives are capable of synthesizing the apo-glucose dehydrogenase (apo-GDH) but not the cofactor, pyrroloquinoline quinone (PQQ), which is essential for the formation of the holoenzyme (3, 5). When PQQ is present in the medium, the holoenzyme is reconstituted and then E. coli is capable of oxidizing glucose to gluconate (7). It has also been reported that the expression of PQQ synthase genes in E. coli resulted in GDH activity in the absence of exogenous PQQ (13). Therefore, we tried to convert D-glucose to 2KDG via D-gluconate by using recombinant E. coli harboring the cloned GADH gene in the presence of PQQ. MATERIALS AND METHODSBacterial strains, media, and culture conditions. E. cypripedii ATCC ...

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“…The LS 3 band was obscured by a band at 1515 cm Ϫ1 assigned as 38 (36). When the temperature was lowered to 90 K, both enzymes exhibited similar RR spectra, with 2 , 3 , and 10 at 1577, 1507, and 1642 cm Ϫ1 , respectively, characteristic of a 6cLS heme. The electronic absorption spectra of the reduced CDH proteins (Fig.…”

Section: Engineering Novel Heme Ligation In Cellobiose Dehydrogenasementioning

confidence: 95%

“…The CDH gene from the white rot fungus Phanerochaete chrysosporium has been cloned and sequenced (2,3), revealing a full-length protein of 755 amino acids partitioned into a cytochrome domain (residues ) and a flavodehydrogenase domain (residues 216 -755) connected by a 25-residue peptide linker. A flavin adenine dinucleotide cofactor is bound to the flavoprotein domain, whereas the cytochrome domain contains a 6-coordinate low spin (6cLS) Fe-protoporphyrin IX (4,5).…”

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confidence: 99%

Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase

Rotsaert

Hällberg

Vries

et al. 2003

Journal of Biological Chemistry

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Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N ␦1 and N ⑀2 nitrogen atoms, rather than the typical N ⑀2 /N ⑀2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N ␦1 /N ⑀2 -coordinated protoporphyrin IX iron.

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Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium

Cited by 62 publications

References 32 publications

Structural Basis of Proteolytic Activation of l-Phenylalanine Oxidase from Pseudomonas sp. P-501

Structural Basis of Proteolytic Activation of l-Phenylalanine Oxidase from Pseudomonas sp. P-501

Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase

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