Development of new disposable NADH biosensors based on NADH oxidase

Creanga, Carmen; Murr, Nabil El

doi:10.1016/j.jelechem.2010.11.030

Cited by 18 publications

(7 citation statements)

References 43 publications

(79 reference statements)

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“…For example, NADH oxidase from Thermus thermophilus showed detectable activity, albeit reduced by about 2 orders of magnitude, in the absence of flavin adenine mononucleotide, which is an essential electron acceptor. 48 Similarly, the metalloenzyme cytidine 3′,5′-cyclic monophosphate phospho-diesterase can catalyze the hydrolysis reaction with no divalent metal cofactor present. 49 For these other enzymes, it is likely that a less optimal pathway has been found to facilitate the reaction.…”

Section: Discussionmentioning

confidence: 99%

Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer

et al. 2014

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X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na+ or K+ coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing 32P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis.

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Section: Discussionmentioning

confidence: 99%

Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer

et al. 2014

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“…The most likely mechanism of NADH oxidation in the presence of free exogenous flavin cofactors is shown in Figure 5, where the presence of flavin cofactor stimulates the oxidase activity of the enzyme. Since free flavin mediates the electron transfer from flavin bound to the enzyme to oxygen [16]:…”

Section: Resultsmentioning

confidence: 99%

“…Tt-NOX presents high chemical and thermal stability, broad-range pH activity, and it can accept NADH or NADPH as substrates [3,10]. Thus, Tt-NOX has successfully been applied to cofactor regeneration systems in the kinetic resolution of secondary alcohols [10] and the selective oxidation of glycerol to yield 1,3-dihydroxyacetone [11,12], in the in situ production of H 2 O 2 coupled to immobilized preparation of peroxidase from horseradish to oxidize pollutants in aqueous solutions [13], in the activation of Pt IV and Ru II chemotherapeutic agents [14], and in biosensors [15,16,17,18,19,20].…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization and Structural Analysis of NADH Oxidase Mutants from Thermus thermophilus HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability

et al. 2019

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The Thermus thermophilus strain HB27 NADH-oxidase (Tt27-NOX) catalyzes the oxidation of nicotinamide adenine dinucleotide (NAD(P)H) by reducing molecular oxygen to hydrogen peroxide in a two-electron transfer mechanism. Surprisingly, Tt27-NOX showed significant differences in catalytic properties compared to its counterpart from the strain HB8 (Tt8-NOX), despite a high degree of sequence homology between both variants. The sequence comparison between both enzymes revealed only three divergent amino acid residues at positions 166, 174, and 194. Motivated with these findings, in this work we performed mutagenesis experiments in the former three positions to study the specific role of these residues in the catalytic properties and thermostability of Tt27-NOX. We subjected five mutants, along with the wild-type enzyme, to biochemical characterization and thermal stability studies. As a result, we identified two more active and more thermostable variants than any Tt8-NOX variant reported in the literature. The most active and thermostable variant K166/H174/Y194 retained 90% of its initial activity after 5 h at pH 7 and 80 °C and an increase in melting temperature of 48.3 °C compared with the least active variant K166/R174/Y194 (inactivated after 15 min of incubation). These results, supported by structural analysis and molecular dynamics simulation studies, suggest that Lys at position 166 may stabilize the loop in which His174 is located, increasing thermal stability.

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“…[3][4][5][6][7] Particularly, the electrochemical methods have attracted much attention due to the possibility to make simple and low cost sensors with reasonable sensitivity. [8][9][10] Nevertheless, the direct electrochemical reaction gives several problems including fairly large overpotentials for both reduction and oxidation, deactivation of electrodes, and formation of undesirable forms such as NAD 2 dimer and inactive 1,6-NADH. [11][12][13] To overcome the drawbacks, indirect electrochemical regeneration of NADH was developed by using various metal complexes or NAD(H)-accepting oxidoreductase enzymes.…”

Section: )mentioning

confidence: 99%

“…[11][12][13] To overcome the drawbacks, indirect electrochemical regeneration of NADH was developed by using various metal complexes or NAD(H)-accepting oxidoreductase enzymes. [4][5][6]8,[15][16][17][18][19][20][21][22][23][24][25] The enzyme-catalyzed generation systems are of great interest and most frequently studied with formate dehydrogenase, 6,15) glucose-6-phosphate dehydrogenase, 16) lipoamide dehydrogenase, 17) and diaphorase. [18][19][20][21][22][23][24][25] Among them, diaphorase (DI) has been most commonly used because of its thermal stability, insensitivity to dioxygen, and the high rates for enzymatic reaction.…”

Section: )mentioning

confidence: 99%

Simple Preparation of Diaphorase/Polysiloxane Viologen Polymer Modified Electrode for Sensing NAD and NADH

Song¹,

Hong²,

Nagarale³

et al. 2011

Journal of Electrochemical Science and Technology

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:Nicotinamide adenine dinucleotide, NAD + , and its reduced form, NADH, play important roles as coenzymes in many enzymatic reactions. Electrochemical methods for NAD + or NADH detection or generation are drawn attention because it can provide the simple and low cost platform with fairly good sensitivity. In this study, the polysiloxane viologen polymer/diaphorase/hydrophilic polyurethane (PSV/DI/HPU) modified electrodes were simply prepared and demonstrated for bio-electrocatalytic NAD + sensors. The electrodes were co-immobilized with diaphorase and polysiloxane viologen polymer as an electron mediator followed by the overcoating with HPU membrane. The mixture of the enzyme and the electron mediator was well stabilized within HPU membrane and exhibited good reversibility and stability. The sensitivity was 0.2 nA·µM −1 and the detection limit was 28 µM with a response time of 50 s (t 90% ). The capability for NADH sensor was also observed on the PSV/DI/HPU electrode.

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Development of new disposable NADH biosensors based on NADH oxidase

Cited by 18 publications

References 43 publications

Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer

Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer

Functional Characterization and Structural Analysis of NADH Oxidase Mutants from Thermus thermophilus HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability

Simple Preparation of Diaphorase/Polysiloxane Viologen Polymer Modified Electrode for Sensing NAD and NADH

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