Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis

Harada, Hiromi; Onoda, Akira; Uchihashi, Takayuki; Watanabe, Hiroki; Sunagawa, Naoki; Samejima, Masahiro; Igarashi, Kiyohiko; Hayashi, Takashi

doi:10.1039/c7sc01672g

Cited by 29 publications

(26 citation statements)

References 39 publications

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“…Structures of the separate domains of P. chrysosporium CDH ( Pc CDH) were published in the early 2000s (CYT CDH : 1.9 Å, PDB 1D7C, (Hallberg et al 2000 ); DH CDH : 1.5 Å, PDB 1KDG, (Hallberg et al 2002 )), while structures of intact CDH were published only recently ( Neurospora crassa CDH IIA: 2.9 Å, PDB 4QI7; Myriococcum thermophilum CDH: 3.2 Å, PDB 4QI6), due to the difficulties in obtaining suitable crystals of the flexible proteins (Tan et al 2015 ). Harada et al recently confirmed the domain movement by using atomic force microscopy (Harada et al 2017 ). This conformational change and the mobility of the two domains are of particular importance for the electron transfer chain described below.…”

Section: Introductionmentioning

confidence: 90%

Multiplicity of enzymatic functions in the CAZy AA3 family

Sützl

Laurent

Abrera

et al. 2018

Appl Microbiol Biotechnol

113

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The CAZy auxiliary activity family 3 (AA3) comprises enzymes from the glucose-methanol-choline (GMC) family of oxidoreductases, which assist the activity of other AA family enzymes via their reaction products or support the action of glycoside hydrolases in lignocellulose degradation. The AA3 family is further divided into four subfamilies, which include cellobiose dehydrogenase, glucose oxidoreductases, aryl-alcohol oxidase, alcohol (methanol) oxidase, and pyranose oxidoreductases. These different enzymes catalyze a wide variety of redox reactions with respect to substrates and co-substrates. The common feature of AA3 family members is the formation of key metabolites such as H2O2 or hydroquinones, which are required by other AA enzymes. The multiplicity of enzymatic functions in the AA3 family is reflected by the multigenicity of AA3 genes in fungi, which also depends on their lifestyle. We provide an overview of the phylogenetic, molecular, and catalytic properties of AA3 enzymes and discuss their interactions with other carbohydrate-active enzymes.

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Section: Introductionmentioning

confidence: 90%

Multiplicity of enzymatic functions in the CAZy AA3 family

Sützl

Laurent

Abrera

et al. 2018

Appl Microbiol Biotechnol

113

View full text Add to dashboard Cite

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“…The biggest contribution to binding comes from the electrostatic interaction of a haem propionate‐A group interacts with an Arg residue on the dehydrogenase domain on the side of the substrate channel . High‐speed atomic force microscopy was used to visualize the dynamic domain motion of P. chrysosporium CDH . An flip‐flop motion was observed involving domain‐domain association in the presence of the substrate cellobiose and subsequent dissociation, whereas the two domains were immobile in the absence of substrate.…”

Section: Direct Electrochemistry Of Flavocytochromesmentioning

confidence: 99%

Direct Electron Transfer of Enzymes Facilitated by Cytochromes

Ludwig

2018

ChemElectroChem

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The direct electron transfer (DET) of enzymes has been utilized to develop biosensors and enzymatic biofuel cells on micro‐ and nanostructured electrodes. Whereas some enzymes exhibit direct electron transfer between their active‐site cofactor and an electrode, other oxidoreductases depend on acquired cytochrome domains or cytochrome subunits as built‐in redox mediators. The physiological function of these cytochromes is to transfer electrons between the active‐site cofactor and a redox partner protein. The exchange of the natural electron acceptor/donor by an electrode has been demonstrated for several cytochrome carrying oxidoreductases. These multi‐cofactor enzymes have been applied in third generation biosensors to detect glucose, lactate, and other analytes. This review investigates and classifies oxidoreductases with a cytochrome domain, enzyme complexes with a cytochrome subunit, and covers designed cytochrome fusion enzymes. The structurally and electrochemically best characterized proponents from each enzyme class carrying a cytochrome, that is, flavoenzymes, quinoenzymes, molybdenum‐cofactor enzymes, iron‐sulfur cluster enzymes, and multi‐haem enzymes, are featured, and their biochemical, kinetic, and electrochemical properties are compared. The cytochromes molecular and functional properties as well as their contribution to the interdomain electron transfer (IET, between active‐site and cytochrome) and DET (between cytochrome and electrode) with regard to the achieved current density is discussed. Protein design strategies for cytochrome‐fused enzymes are reviewed and the limiting factors as well as strategies to overcome them are outlined.

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“…In ar ecent paper,H arada et al exploitedh ighspeed atomicf orce microscopy to measure in operando the conformational changes of the cellobiosed ehydrogenase immobilized on flat gold surfaces functionalized with heme groups. [42] This work sheds light on the mechanism of this multi-domain protein at nanometric scale. The highly specialized infrastructure required for these studies makes that spatial resolution studies monitoring the catalytic activity of the immobilized enzymes are dominated by fluorescence spectroscopy;aworldwide and highly accessible technique.…”

Section: Single-particle Reaction Kinetics Of Immobilized Enzymesmentioning

confidence: 90%

“…In a recent paper, Harada et al. exploited high‐speed atomic force microscopy to measure in operando the conformational changes of the cellobiose dehydrogenase immobilized on flat gold surfaces functionalized with heme groups . This work sheds light on the mechanism of this multi‐domain protein at nanometric scale.…”

Section: Single‐particle Reaction Kinetics Of Immobilized Enzymesmentioning

confidence: 99%

Single‐Particle Studies to Advance the Characterization of Heterogeneous Biocatalysts

et al. 2018

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Immobilized enzymes have been widely exploited because they work as heterogeneous biocatalysts, allowing their recovery and reutilization and easing the downstream processing once the chemical reactions are completed. Unfortunately, we suffer a lack of analytical methods to characterize those heterogeneous biocatalysts at microscopic and molecular levels with spatio‐temporal resolution, which limits their design and optimization. Single‐particle studies are vital to optimize the performance of immobilized enzymes in micro/nanoscopic environments. In this Concept article, we review different analytical techniques that address single‐particle studies to image the spatial distribution of the enzymes across the solid surfaces, the sub‐particle substrate diffusion, the structural integrity and mobility of the immobilized enzymes inside the solid particles, and the pH and O2 internal gradients. From our view, such sub‐particle information elicited from single‐particle analysis is paramount for the design and fabrication of optimal heterogeneous biocatalyst.

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Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis

Abstract: To visualize the dynamic domain motion of class-I CDH from Phanerochaete chrysosporium (PcCDH) during catalysis using high-speed atomic force microscopy, the apo-form of PcCDH was anchored to a heme-immobilized flat gold surface that can fix the orientation of the CYT domain.

Cited by 29 publications

References 39 publications

Multiplicity of enzymatic functions in the CAZy AA3 family

Multiplicity of enzymatic functions in the CAZy AA3 family

Direct Electron Transfer of Enzymes Facilitated by Cytochromes

Single‐Particle Studies to Advance the Characterization of Heterogeneous Biocatalysts

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