Formate Oxidase, an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family, Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

Doubayashi, Daiju; Ootake, Takumi; Maeda, Yosifumi; Oki, Masaya; Tokunaga, Yuji; Sakurai, Akihiko; Nagaosa, Yukio; Mikami, Bunzo; Uchida, Hiroyuki

doi:10.1271/bbb.110153

Cited by 22 publications

(22 citation statements)

References 30 publications

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“…3 D ). Overall, the 1 H NMR spectrum possesses the same features as reported previously for 8-formyl-FAD isolated from formate oxidase ( 8 ). Taken together with the observed mass difference, we therefore conclude that the 8α-methyl group of FAD was oxidized to a formyl group to yield 8f-FAD.…”

Section: Resultssupporting

confidence: 77%

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Augustín

Toplak

Fuchs

et al. 2018

Journal of Biological Chemistry

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The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

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Section: Resultssupporting

confidence: 77%

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Augustín

Toplak

Fuchs

et al. 2018

Journal of Biological Chemistry

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“…The Lys225 NH 3 + atoms, Gly120/Asn119 peptide bond atoms and FAD-N5 atoms are in the most well ordered region of the structure and have low refined B-factors (<4.5) compared to the overall B-factor of the structure (14.1). A charged side chain interacting with the protein carbonyl appears to be a conserved feature in FAD-dependent oxidoreductases of the GMC oxidoreductase family with an arginine side chain present in glucose oxidase (1GPE15), cellobiose dehydrogenase (1KDG)24, aryl-alcohol dehydrogenase (3FIM)25, 5-hydroxymethylfurfural oxidase (4UDP)26, formate oxidase (3Q9T)27, choline oxidase (2JBV)28 hydroxynitrile lyase (1JU2)29 and a histidine residue in pyranose-2-oxidase (1TZL)30 (Fig. 3b).…”

Section: Resultsmentioning

confidence: 99%

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

Golden

Meilleur

et al. 2017

Sci Rep

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The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.

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“…Although the FAD binding and substrate binding domains are primarily in the N-and C-terminal part of the sequence, respectively, parts of the structure of each domain are insertions in the other domain, as is the case in all known members of this family. A structure based comparison of R135 with other members of the GMC oxidoreductase family using the SALAMI server (http://flensburg.zbh.uni-hamburg.de/∼wurst/salami/) shows that the closest homologues in the PDB are a fungal aryl alcohol oxidase (FAAO) involved in lignin biodegradation (PDB: 3FIM) (Fernández et al, 2009) and a formate oxidase (PDB: 3Q9T) (Doubayashi et al, 2011). There are overall r.m.s.d's of about 3Å between equivalenced C α atoms for 75.6% and 78% of the residues in the R135_50 and FAAO/formate oxidase structures and a sequence identity of 18% and 22%, respectively.…”

Section: Resultsmentioning

confidence: 99%

A Mimivirus Enzyme that Participates in Viral Entry

et al. 2015

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Summary Mimivirus was initially identified as a bacterium because its dense, 125nm-long fibers stained Gram-positive. These fibers probably play a role during the infection of some host cells. The normal hosts of Mimivirus are unknown, but in the laboratory Mimivirus is usually propagated in amoeba. The structure of R135, a major component of the fibrous outer layer of Mimivirus, has been determined to 2Å resolution. The protein's structure is similar to members of the glucose-methanol-cholin oxidoreductase family, which have an N-terminal FAD binding domain and a C-terminal substrate recognition domain. The closest homologue to R135 is an aryl alcohol oxidase that participates in lignin biodegradation of plant cell walls. Thus R135 might participate in the degradation of their normal hosts, including some lignin-containing algae.

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Formate Oxidase, an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family, Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

Cited by 22 publications

References 30 publications

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

A Mimivirus Enzyme that Participates in Viral Entry

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