RESUMO
Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric l-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of -0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of l-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for l-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 µA mM-1 cm-2, and the detection limit was 13.6 µM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of l-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Neoplasias , Agaricales , Detecção Precoce de Câncer , Eletrodos , Fucose , Ouro , HumanosRESUMO
Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase is one of the extensively studied sugar-oxidizing enzymes used as a biocatalyst for biosensors and biofuel cells. A novel pyranose dehydrogenase (CcPDH) derived from the basidiomycete Coprinopsis cinerea is the first discovered eukaryotic PQQ-dependent enzyme. This enzyme carries a b-type cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH); thus, CcPDH is a quinohemoprotein. CcPDH catalyzes the oxidation of various aldose sugars and shows significant activity toward the reverse-chair conformation of pyranoses. Interdomain electron transfer occurs in CcPDH similar to CDH, from the PQQ cofactor in the catalytic domain to the heme b in the cytochrome domain. This enzyme is able to direct electrical communication with electrodes, without artificial electron mediators, thus allowing direct electron transfer (DET)-type bioelectrocatalysis. In this review, we briefly describe recent progress in research on the biochemical discovery of CcPDH and the development of (bio)electrochemical applications (an amperometric biosensor) based on DET reactions.
Assuntos
Técnicas Eletroquímicas/instrumentação , Hemeproteínas/química , Quinonas/química , Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais , EucariotosRESUMO
Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade ß-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.
Assuntos
Citocromos b/química , Eucariotos/enzimologia , Glucose Desidrogenase/metabolismo , Oxirredutases/química , Cofator PQQ/química , Agaricales/enzimologia , Agaricales/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Sítios de Ligação , Desidrogenases de Carboidrato/metabolismo , Catálise , Citocromos b/metabolismo , Transporte de Elétrons , Eucariotos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Modelos Moleculares , Oxirredução , Oxirredutases/metabolismo , Cofator PQQ/metabolismo , Conformação Proteica , Domínios Proteicos , Difração de Raios XRESUMO
In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. CcPDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). CcPDH oxidizes 2-keto-d-glucose (d-glucosone), l-fucose, and rare sugars such as d-arabinose and l-galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes.
Assuntos
Basidiomycota/enzimologia , Biocatálise , Oxirredutases/metabolismo , Cofator PQQ/metabolismo , Arabinose/metabolismo , Fucose/metabolismo , Galactose/metabolismo , Cetoses/metabolismo , Oxirredução , Especificidade por SubstratoRESUMO
NAD(P)-dependent group III alcohol dehydrogenases (ADHs), well known as iron-activated enzymes, generally lose their activities under aerobic conditions due to their oxygen-sensitivities. In this paper, we expressed an extremely thermostable group III ADH from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (PhADH) heterologously in Escherichia coli. When purified from a culture medium containing nickel, the recombinant PhADH (Ni-PhADH) contained 0.85 ± 0.01 g-atoms of nickel per subunit. Ni-PhADH retained high activity under aerobic conditions (9.80 U mg-1), while the enzyme expressed without adding nickel contained 0.46 ± 0.01 g-atoms of iron per subunit and showed little activity (0.27 U mg-1). In the presence of oxygen, the activity of the Fe2+-reconstituted PhADH prepared from the Ni-PhADH was gradually decreased, whereas the Ni2+-reconstituted PhADH maintained enzymatic activity. These results indicated that PhADH with bound nickel ion was stable in oxygen. The activity of the Ni2+-reconstituted PhADH prepared from the expression without adding nickel was significantly lower than that from the Ni-PhADH, suggesting that binding a nickel ion to PhADH in this expression system contributed to protecting against inactivation during the expression and purification processes. Unlike other thermophilic group III ADHs, Ni-PhADH showed high affinity for NAD(H) rather than NADP(H). Furthermore, it showed an unusually high k cat value toward aldehyde reduction. The activity of Ni-PhADH for butanal reduction was increased to 60.7 U mg-1 with increasing the temperature to 95 °C. These findings provide a new strategy to obtain oxygen-sensitive group III ADHs.
Assuntos
Álcool Desidrogenase/genética , Regulação Enzimológica da Expressão Gênica/genética , Oxigênio/metabolismo , Reação em Cadeia da Polimerase , Pyrococcus horikoshii/enzimologia , Álcool Desidrogenase/isolamento & purificação , Álcool Desidrogenase/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
OBJECTIVE: To explore the feasibility and diagnostic accuracy of modified subtraction coronary CT angiography (CCTA) with short breath-holding time in patients who have limited breath-hold capability and severe coronary artery calcification. METHODS: 11 patients with a coronary calcium score >400 underwent CCTA using a modified subtraction protocol. All patients were unable to hold their breath for more than 20 s. Subjective image quality using a four-point scale and the presence of significant (>50%) luminal stenosis were assessed for each calcified or stented segment on both conventional CCTA and modified subtraction CCTA images and compared with invasive coronary angiography (ICA) as the gold standard. RESULTS: The mean breath-holding time was 13.0 ± 0.9 s. A total of 35 calcified or stented coronary segments were evaluated. The average image quality was increased from 2.1 ± 0.9 with conventional CCTA to 3.1 ± 0.7 with subtraction CCTA (p < 0.001). The segment-based diagnostic accuracy for detecting significant stenosis according to ICA revealed an area under the receiver-operating characteristic curve of 0.722 for conventional CCTA and 0.892 for subtraction CCTA (p = 0.036). CONCLUSION: Modified subtraction CCTA allows the breath-holding time to be shortened to <15 s. As compared with conventional CCTA, modified subtraction CCTA showed improvement in image quality and diagnostic accuracy in patients with limited breath-hold capability and severe calcification. ADVANCES IN KNOWLEDGE: Modified subtraction CCTA can improve the diagnostic accuracy in patients with a high calcium score and patients who are unable to perform long breath-holds.
Assuntos
Angiografia Digital , Suspensão da Respiração , Angiografia por Tomografia Computadorizada/métodos , Estenose Coronária/diagnóstico por imagem , Calcificação Vascular/diagnóstico por imagem , Idoso , Artefatos , Estudos de Viabilidade , Feminino , Humanos , Masculino , Valor Preditivo dos Testes , Interpretação de Imagem Radiográfica Assistida por Computador , Sensibilidade e EspecificidadeRESUMO
RATIONALE AND OBJECTIVES: Severe calcifications of the coronary arteries are still a major challenge in coronary computed tomography (CT) angiography (CCTA). Subtraction CCTA using a 320-detector row CT scanner has recently been introduced for patients with severe calcifications. However, the conventional subtraction CCTA method requires a long breath-holding time of approximately 20-40 seconds. This is a major problem in clinical practice because many patients may not be able to perform such a long breath-hold. We explored a modified subtraction CCTA method with a short breath-holding time to overcome this problem. MATERIALS AND METHODS: This study was approved by our institutional review board, and all patients gave written informed consent. A total of 12 patients with a coronary calcium score of >400 were enrolled in this study. All patients were unable to hold their breath for more than 20 seconds. Modified subtraction CCTA was performed using the bolus-tracking method. The acquisition protocol was adjusted so that the mask scan was acquired 10 seconds after the postcontrast scan during a single breath-hold. The subtraction image was obtained by subtracting the mask image data from the postcontrast image data. The breath-holding times were recorded. Enhancement of the coronary arteries in the subtraction images was assessed. Subjective image quality was evaluated in a total of 32 segments using a 4-point scale. RESULTS: The mean breath-holding time was 12.8 ± 0.8 seconds (range, 12-14 seconds). The average CT number in the coronary arteries was 288.6 ± 80.5 Hounsfield units (HU) in the subtraction images. Average image quality was significantly increased from 2.1 ± 0.9 with conventional CCTA to 3.1 ± 0.7 with subtraction CCTA (P < 0.001). With subtraction CCTA, the number of non-diagnostic segments was significantly reduced from 53% to 19% (P = 0.001). CONCLUSIONS: This preliminary study has shown that our modified subtraction CCTA method allows the breath-holding time to be shortened to <15 seconds. This may substantially improve the success rate of subtraction CCTA by reducing artifacts and allowing this technique to be applied to patients who are unable to perform a long breath-hold.
Assuntos
Suspensão da Respiração , Angiografia por Tomografia Computadorizada/métodos , Angiografia Coronária/métodos , Técnica de Subtração , Idoso , Artefatos , Vasos Coronários/diagnóstico por imagem , Feminino , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of l-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters.
Assuntos
Concentração de Íons de Hidrogênio , Biocatálise , Técnicas Eletroquímicas , Transporte de ElétronsRESUMO
The basidiomycete Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH; EC 1.1.99.18) from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.
Assuntos
Agaricales/enzimologia , Metabolismo dos Carboidratos , Oxirredutases/química , Oxirredutases/metabolismo , Cofator PQQ/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Eletroquímica , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The purpose of this study was to explore the feasibility of subtraction coronary computed tomography angiography (CCTA) by second-generation 320-detector row CT in patients with severe coronary artery calcification using invasive coronary angiography (ICA) as the gold standard. This study was approved by the institutional board, and all subjects provided written consent. Twenty patients with calcium scores of >400 underwent conventional CCTA and subtraction CCTA followed by ICA. A total of 82 segments were evaluated for image quality using a 4-point scale and the presence of significant (>50 %) luminal stenosis by two independent readers. The average image quality was 2.3 ± 0.8 with conventional CCTA and 3.2 ± 0.6 with subtraction CCTA (P < 0.001). The percentage of segments with non-diagnostic image quality was 43.9 % on conventional CCTA versus 8.5 % on subtraction CCTA (P = 0.004). The segment-based diagnostic accuracy for detecting significant stenosis according to ICA revealed an area under the receiver operating characteristics curve of 0.824 (95 % confidence interval [CI], 0.750-0.899) for conventional CCTA and 0.936 (95 % CI 0.889-0.936) for subtraction CCTA (P = 0.001). The sensitivity, specificity, positive predictive value, and negative predictive value for conventional CCTA were 88.2, 62.5, 62.5, and 88.2 %, respectively, and for subtraction CCTA they were 94.1, 85.4, 82.1, and 95.3 %, respectively. As compared to conventional, subtraction CCTA using a second-generation 320-detector row CT showed improvement in diagnostic accuracy at segment base analysis in patients with severe calcifications.
Assuntos
Angiografia Digital/métodos , Angiografia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Estenose Coronária/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Tomografia Computadorizada Multidetectores/métodos , Calcificação Vascular/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Estudos Prospectivos , Interpretação de Imagem Radiográfica Assistida por Computador , Reprodutibilidade dos Testes , Índice de Gravidade de DoençaRESUMO
A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucose Desidrogenase/metabolismo , Cofator PQQ/metabolismo , Pseudomonas/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Glucose Desidrogenase/genética , Dados de Sequência Molecular , FilogeniaRESUMO
An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.
Assuntos
Citocromos c/química , Microscopia de Força Atômica , Adsorção , Animais , Eletroquímica , Eletrodos , Ácidos Graxos/química , Ouro/química , Modelos Moleculares , Conformação Proteica , Compostos de Sulfidrila/química , Fatores de TempoRESUMO
Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.
Assuntos
Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Aminas/farmacologia , Cofator PQQ/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Pseudomonas putida/enzimologiaRESUMO
Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.
Assuntos
Bases de Dados de Proteínas , Oxirredutases/química , Cofator PQQ/química , Sequência de Aminoácidos , Sequência de Bases , Basidiomycota/enzimologia , Calorimetria , Primers do DNA , Dados de Sequência Molecular , Oxirredutases/genética , Filogenia , Pichia/genética , Homologia de Sequência de AminoácidosRESUMO
A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH.