The role of double covalent flavin binding in chito-oligosaccharide oxidase from <i>Fusarium graminearum</i>

Heuts, Dominic P. H. M.; Winter, Remko T.; Damsma, Gerke E.; Janssen, Dick B.; Fraaije, Marco W.

doi:10.1042/bj20071591

Cited by 54 publications

(57 citation statements)

References 29 publications

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“…Such a drastic change in the mechanism of hydride transfer most likely arises from either an increased conformational freedom or a rearrangement of the position of the flavin isoalloxazine ring acting as the hydride ion acceptor with respect to the choline alkoxide acting as the hydride donor, which necessarily results from the lack of the flavin covalent attachment to the protein moiety. 3 This, in turn, establishes that the covalent linkage in flavin-dependent enzymes, besides stabilizing protein structure (20 -22), preventing the loss of loosely bound flavin cofactors (23), modulating the redox potential of the flavin (20,(23)(24)(25)(26)(27), and facilitating electron transfer reactions (28) as amply demonstrated from studies of enzymes other than choline oxidase, may also be important for the optimal positioning of the flavin in those enzymes where hydride ions are transferred through tunneling mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…Glucooligosaccharide oxidase (15,16), hexose oxidase (17), and berberine bridge enzyme (18,19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20 -22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20,(23)(24)(25)(26)(27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.…”

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confidence: 99%

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Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

Quaye¹,

Cowins

Gadda

2009

Journal of Biological Chemistry

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A number of enzymes, including dehydrogenases (1-3), monooxygenases (4 -7), halogenases (8 -11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18,19) The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31-33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35,36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12,38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39 -45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46 -48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofac-

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

Quaye¹,

Cowins

Gadda

2009

Journal of Biological Chemistry

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“…1c and Supplementary Fig. 2 online), thus providing an additional example for the recently discovered group of bi-covalently flavinylated enzymes [6][7][8][9][10] . The active site cavity close to the cofactor is lined by mainly hydrophobic residues with the only notable exceptions being Tyr106, Thr358, Asn390, Glu417 and His459 (Fig.…”

Section: Berberine Bridge Enzyme (Bbe) Catalyzes the Conversion Of (Smentioning

confidence: 99%

A concerted mechanism for berberine bridge enzyme

et al. 2008

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“…4. Steady-state Kinetics-Due to significant changes in the affinity of substrates to protein variants with a modified FAD attachment observed in both GOOX and ChitO (11,17) we analyzed the steady-state kinetics of WT BBE in comparison with both mutant proteins over a substrate concentration range of 2-350 M ( Table 1). The highest value represents about 100 times the reported K m for WT BBE (26).…”

Section: Optimization Of Expression and Proteinmentioning

confidence: 99%

“…prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic func-tioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about ϩ130 mV, which is among the highest potentials reported for flavoenzymes.…”

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confidence: 99%

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Winkler

Motz

Riedl

et al. 2009

Journal of Biological Chemistry

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Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8␣-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8␣-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8␣-histidylation (either to N1 or to N3), 8␣-O-tyrosylation, 8␣-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX) 4 from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8␣-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7-9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12)...

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The role of double covalent flavin binding in chito-oligosaccharide oxidase from Fusarium graminearum

Cited by 54 publications

References 29 publications

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

A concerted mechanism for berberine bridge enzyme

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

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