Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

Caldinelli, Laura; Iametti, Stefania; Barbiroli, Alberto; Bonomi, Francesco; Piubelli, Luciano; Ferranti, Pasquale; Picariello, Gianluca; Pilone, Mirella S.; Pollegioni, Loredano

doi:10.1074/jbc.m403489200

Cited by 27 publications

(48 citation statements)

References 29 publications

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“…This indicates that re-establishing the proper protein-cofactor interaction during the refolding process was somewhat impaired. Noteworthy, the same results were also recently observed in the case of D-amino acid oxidase, a FAD-containing flavoprotein carrying a non-covalently bound flavin (24).…”

Section: Fig 3 Results Of Titration Of Wild-type (A) and H69a (B) Csupporting

confidence: 57%

“…At first, both proteins were titrated with ANS in the presence of various urea concentrations, allowing determination of the change in fluorescence emission intensity at saturating ANS concentration (⌬F), the apparent K d for ANS binding, and the ratio between these two parameters (⌬F/K d ) (24). Values of the apparent K d for ANS binding to wild-type CO at increasing urea concentrations remained quite constant (135 Ϯ 20 M), whereas the parameter ⌬F/K d varied with the urea concentration, attaining a maximum at 4 M urea (Fig.…”

Section: Fig 1 Equilibrium Denaturation Curves Of Wild-type (Circlementioning

confidence: 99%

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Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Caldinelli

Iametti

Barbiroli

et al. 2005

Journal of Biological Chemistry

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Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His 69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (ϳ3 versus ϳ5 M, respectively), and the mutant protein had a melting temperature ϳ10 -15°C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 M urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.Bacterial cholesterol oxidase (CO, EC 1.1.3.6) is a monomeric bifunctional FAD-containing flavoenzyme that catalyzes the oxidation of 3␤-hydroxysteroids and the isomerization of the intermediate, ⌬ 5-6 -ene-3␤-ketosteroid (cholest-5-en-3-one) to produce ⌬ 3-4 -ene-3␤-ketosteroid (cholest-4-en-3-one). Bacteria that produce CO can be classified into two classes: non-pathogenic (e.g. Streptomyces and fast-growing Mycobacteria; these bacteria utilize cholesterol as their carbon source) and pathogenic bacteria (e.g. Rhodococcus equi and slow-growing Mycobacteria). Pathogenic bacteria require CO for infection of the host macrophage probably because of its ability to alter the physical structure of the membrane by converting cholesterol into cholest-4-en-3-one.The crystal structure of COs belonging to two distinct structural classes (1) have been solved by the Vrielink laboratory (2-4). Members classified as type I, such as those from Streptomyces sp. SA-COO and R. equi (5) have their cofactor tightly but non-covalently bound to the enzyme. A second CO from Brevibacterium sterolicum classified as type II, is completely different. In this enzyme the cofactor is covalently linked to the protein via a bond between the 8-methyl group of the isoalloxazine ring and the ND1 atom of His 69 .Although COs of class I an...

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Section: Fig 3 Results Of Titration Of Wild-type (A) and H69a (B) Csupporting

confidence: 57%

Section: Fig 1 Equilibrium Denaturation Curves Of Wild-type (Circlementioning

confidence: 99%

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Caldinelli

Iametti

Barbiroli

et al. 2005

Journal of Biological Chemistry

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“…Although A300 is conserved in most enzymes, its replacement with Thr, as (34). However, after reactivation with cysteine and FAD, the variant recovered some activity, suggesting that the N302S variant may be in a partially folded and expanded dimeric state, like unfolding intermediates previously described for other flavoproteins (6,23). This partially unfolded form may partly recover the compact dimer structure after reactivation.…”

Section: Discussionmentioning

confidence: 72%

Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

2011

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Water-forming NADH oxidases (encoded by noxE, nox2, or nox) are flavoproteins generally implicated in the aerobic survival of microaerophilic bacteria, such as lactic acid bacteria. However, some natural Lactococcus lactis strains produce an inactive NoxE. We examined the role of NoxE in the oxygen tolerance of L. lactis in the rich synthetic medium GM17. Inactivation of noxE suppressed 95% of NADH oxidase activity but only slightly affected aerobic growth, oxidative stress resistance, and NAD regeneration. However, noxE inactivation strongly impaired oxygen consumption and mixed-acid fermentation. We found that the A303T mutation is responsible for the loss of activity of a naturally occurring variant of NoxE. Replacement of A303 with T or G or of G307 with S or A by site-directed mutagenesis led to NoxE aggregation and the total loss of activity. We demonstrated that L299 is involved in NoxE activity, probably contributing to positioning flavin adenine dinucleotide (FAD) in the active site. These residues are part of the strongly conserved sequence LA(T)XXA XXXG included in an alpha helix that is present in other flavoprotein disulfide reductase (FDR) family flavoproteins that display very similar three-dimensional structures.Water-forming NADH oxidases (Nox, NoxE, or Nox2) are flavoproteins involved in the aerobic growth and survival of lactic acid bacteria (LAB) under fermentation conditions (7). Their inactivation in several streptococci strongly reduces the aerobic growth. Inactivation of nox2 in Streptococcus pneumoniae and Streptococcus agalactiae reduces growth in aerated media by 80% (37, 38). The growth defect of the S. agalactiae mutant has been attributed to a defect in fatty acid production resulting from nonproduction of the fatty acid precursor acetyl-coenzyme A (CoA) due to strictly homolactic fermentation (37). Nox2 inactivation in Streptococcus mutans also leads to strict homolactic fermentation on glucose; however, it does not affect growth on glucose but greatly reduces growth on mannitol (11). In Streptococcus pyogenes, Nox2 inactivation completely abolishes aerobic growth under carbon limitation conditions and increases sensitivity to the superoxide-generating agent paraquat (methyl viologen). This growth defect has been attributed to hydrogen peroxide (H 2 O 2 ) accumulation (10). NoxE is the principal enzyme displaying NADH oxidase (NOX) activity in Lactococcus lactis (95% of the NADH oxidase activity of L. lactis TIL46), a LAB widely used in industrial milk fermentations and generally considered a model LAB. However, NoxE inactivation in L. lactis reduces the growth rate in milk by only 20%, although it strongly impairs oxygen consumption (34). Moreover, we isolated two natural L. lactis strains without detectable NADH oxidase activity from matured cheese (34), suggesting that NoxE is not important for L. lactis survival in dairy media. In one natural NADH oxidase-negative strain, the noxE gene was complete but the enzyme appeared to be inactive. Several water-forming NADH oxidases from...

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“…Surprisingly, it has been reported that DAAO can assemble properly in the cytosol and thus that the catabolic utilization of Damino acids is also active in the cytosol of C. boidinii [31] and H. polymorpha cells [32]. In contrast, we recently hypothesized that the folding intermediate identified for RgDAAO possesses the properties (e.g., it lacks the catalytic activity and the characteristic tertiary structure of the native enzyme but has a significant secondary structure and retains flavin binding) proposed for the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane [50,51]. Biochemical properties of fungal DAAOs.…”

Section: Daaos From Fungimentioning

confidence: 90%

Physiological functions of D-amino acid oxidases: from yeast to humans

Pollegioni

Piubelli

Sacchi

et al. 2007

Cell. Mol. Life Sci.

319

304

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D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.

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Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

Cited by 27 publications

References 29 publications

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Dissecting the Structural Determinants of the Stability of Cholesterol Oxidase Containing Covalently Bound Flavin

Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

Physiological functions of D-amino acid oxidases: from yeast to humans

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