Deflavination of flavo-oxidases by nucleophilic reagents

Zlateva, Theodora; Boteva, Raina; Filippi, Bruno; Veenhuis, Marten; Klei, Ida J. van der

doi:10.1016/s0167-4838(01)00233-3

Cited by 10 publications

(10 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, this position between Trp47 and the flavin cofactor can be achieved only in the dimeric form of the enzyme [9]. In accordance with previously published CD spectra of the flavin oxidases [42], the pronounced peak at 265 nm may result from an asymmetric environment of the tightly bound flavin cofactor and/or Trp47 in the active site of the enzyme, and Trp52. The small negative ellipticity at 286 nm corresponds to the signal of tryptophan residues.…”

Section: Resultssupporting

confidence: 73%

“…A slight decrease in the positive ellipticity at ≈ 265 nm in the presence of perchlorate anions, i.e. a decrease in the asymmetry of the tryptophan residue and/or the flavin cofactor in the active site, may result from dissociation of the flavin cofactor in the presence of nucleophilic agents [42]. A 24 h dialysis of NADH oxidase in the presence of 2 m perchlorate anions did not cause dissociation of the flavin cofactor (data not shown).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions

Žoldák

Sprinzl

Sedlák

2003

European Journal of Biochemistry

View full text Add to dashboard Cite

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H 2 PO 4 -, SO 4 2-, CH 3 COO -, Cl -, Br -, I -, ClO 4 -, SCN -) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.Keywords: activation; conformational dynamics; flavoproteins; NADH oxidase; Thermus thermophilus.The native conformation of an enzyme is produced by the complex interaction of van der Waals interactions, hydrogen bonds and ionic interactions. These interactions produce stability of the enzyme under physiological conditions and prevent deleterious conformational changes from perturbations in the environment that would cause deactivation. These interactions, however, must not result in protein rigidity because the enzyme active site requires flexibility for optimal catalytic function. The balance of these two tendencies is sensitively adjusted for the physiological conditions at which the enzyme works. Examples of such adjustments are enzymes from hyperthermophiles and psychrophiles which have optimal activity at high (> 80°C) and low (< 20°C) temperatures, respectively [1,2]. Enzymes from thermophiles are almost inactive at room temperature because of polypeptide and side chain rigidity induced by higher-order interactions within secondary and tertiary structures. Psychrophilic enzymes are inactive at room temperature because the high flexibility of their polypeptide and side chains results in partial/local or complete unfolding of the tertiary structure. Modulation of the balance between the rigidity and flexibility of the polypeptide and side chains can be achieved by changing the solvent properties. Stabilization of psychrophilic enzymes without affecting their activity, or activation of thermophilic enzymes without affecting their stability, is interesting for both basic and applied...

show abstract

Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions

Žoldák

Sprinzl

Sedlák

2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…In case of GOX, release of FAD did not change the conformation of the protein and its enzyme activity was fully recovered upon addition of excess FAD, suggesting that FAD had re‐associated to the protein. By contrast, the conformation of AO had drastically changed following FAD release and the dissociation of FAD from AO appeared to be irreversible [55].…”

Section: Ao Structurementioning

confidence: 95%

Alcohol oxidase: A complex peroxisomal, oligomeric flavoprotein

Ozimek

Veenhuis

Klei

2005

FEMS Yeast Research

View full text Add to dashboard Cite

Alcohol oxidase (AO) is the key enzyme of methanol metabolism in methylotrophic yeast species. It catalyses the first step of methanol catabolism, namely its oxidation to formaldehyde with concomitant production of hydrogen peroxide. In its mature active form, AO is a molecule of high molecular mass (600 kDa) that consists of eight identical subunits, each of which carry one non-covalently bound flavin adenine nucleotide (FAD) molecule as the prosthetic group. In vivo, the protein is compartmentalized into special cell organelles, termed peroxisomes. AO is an abundant protein and its synthesis is strictly regulated by repression/derepression and induction mechanisms that occur at the transcriptional level. Various aspects of its sorting and assembly/activation render AO a unique protein. Recent developments of AO synthesis, sorting and assembly/activation are highlighted in this paper.

show abstract

“…Chloride is less chaotropic and therefore less effective in removal of the flavin [111]. Stronger chaotropes such as cyanide, cyanate and thiocyanate have been used as well, but with these nucleophilic agents significant conformational perturbations preventing holoprotein reconstitution may occur [109,112,113].…”

Section: Conventional Methodsmentioning

confidence: 99%

Deflavination and reconstitution of flavoproteins

Hefti¹,

Vervoort²,

Berkel³

2003

European Journal of Biochemistry

116

104

View full text Add to dashboard Cite

Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apoprotein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent accessibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavindeficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification.

show abstract

Deflavination of flavo-oxidases by nucleophilic reagents

Cited by 10 publications

References 29 publications

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions

Alcohol oxidase: A complex peroxisomal, oligomeric flavoprotein

Deflavination and reconstitution of flavoproteins

Contact Info

Product

Resources

About