Structure and mechanism of para‐hydroxybenzoate hydroxylase

Entsch, Barrie; Berkel, W.J.H. van

doi:10.1096/fasebj.9.7.7737455

Cited by 208 publications

(216 citation statements)

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“…1), and the isoalloxazine ring is not directly solvent-accessible. A similar situation was also observed in DAAO (10, 11), whereas in p-hydroxybenzoate hydroxylase the flavin benzene ring is exposed to the bulk solvent, allowing the flavin to adopt two different conformations (36). The large majority of the potential FAD hydrogen bonds are formed with the protein residues, thus resulting in a tight net as shown in Fig.…”

Section: Resultssupporting

confidence: 72%

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Mörtl¹,

Diederichs

Welte

et al. 2004

Journal of Biological Chemistry

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Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 Å, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as D-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR 2 subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or D-alanine as carbon or nitrogen sources.Glycine oxidase (GO, 1 EC 1.4.3.19) is a flavoprotein consisting of four identical subunits (369 residues each) and containing one molecule of noncovalently bound FAD per 42-kDa protein molecule (1, 2). GO catalyzes a reaction similar to that of D-amino acid oxidase (DAAO, EC 1.4.3.3), a paradigm of the dehydrogenase-oxidase class of flavoproteins (for a recent review see Ref.3). Both enzymes catalyze the oxidative deamination of amino acids to yield the corresponding ␣-imino acids and, after hydrolysis, ␣-keto acids, ammonia (or primary amines), and hydrogen peroxide. Both enzymes show a high pK a for flavin N-3H ionization, do not bind covalently the FAD cofactor, and react readily with sulfite (1-3), but they differ in substrate specificity. In addition to neutral D-amino acids (e.g. D-alanine, D-proline, etc., which are also good substrates of DAAO), GO catalyzes the oxidation of primary and secondary amines (e.g. glycine, sarcosine, etc.) partially sharing the substrate specificity with monomeric sarcosine oxidase (MSOX, EC 1.5.3.1), an enzyme that catalyzes the oxidative demethylation of sarcosine to yield glycine, formaldehyde, and hydrogen peroxide (4). According to investigations of the substrate specificity and of the binding properties, the GO active site seems to preferentially accommodate amines of a small size, such as glycine and sarcosine (1, 2). GO follows a ternary complex sequential mechanism with glycine, sarcosine, and D-proline as substrates in which the rate of product dissociat...

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Section: Resultssupporting

confidence: 72%

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Mörtl¹,

Diederichs

Welte

et al. 2004

Journal of Biological Chemistry

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“…The hydrogen bonds between HPA O4 and two side chains (Ser-146 and His-120) could favor preferential binding of the deprotonated form of the substrate (Fig. 5b), which would be more reactive than the protonated form in promoting the electrophilic attack by the C4a-hydroperoxyflavin (25) (Fig. 6).…”

Section: Discussionmentioning

confidence: 99%

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

Alfieri

Fersini

Ruangchan

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

133

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p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADHdependent FMN reductase and a monooxygenase (C 2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C 2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C 2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C 2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-Å radius and a 29-Å 3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. F lavoprotein monooxygenases use dioxygen to insert an oxygen atom into a substrate and have been found to be involved in a wide variety of biological reactions (1-3). The fundamental property of these enzymes is their ability to promote formation and stabilization of the C4a-hydroperoxyflavin (Fig. 1a) resulting from the reaction of the protein-bound reduced flavin with dioxygen. This key intermediate donates an oxygen atom to the substrate, generating the unstable C4a-hydroxyflavin that eliminates one molecule of water to yield oxidized flavin (5). Understanding the structural bases governing functional properties of monooxygenases is crucial to address one of the most fascinating issues in flavoenzymology: the ability of flavoenzymes to differentially react with molecular oxygen.In recent years, a new group of flavoprotein monooxygenases has been identified. These enzymes consist of two components: a reductase generating reduced flavin and a hydroxylase using reduced flavin to catalyze substrate monooxygenation (6). phydroxyphenylacetate hydroxylase from Acinetobacter baumannii catalyzes hydroxylation of p-hydroxyphenylacetate (HPA) to 3,4-dihydroxyphenylacetate (Fig. 1a). HPA hydroxylase has unusual features in both sequence and catalysis. The smaller reductase component of HPA hydroxylase (C 1 ) performs HPA-stimulated NADH-dependent reduction of free FMN, which is subsequently transferred to the larger monooxygenase component (C 2 ) and used for reaction with dioxygen and HPA monooxygenation (Fig. 1b). Specificity for FMN is conferred by C 1 , whereas C 2 works equally well with both reduced FMN (FMNH Ϫ ; Fig. 1a) and reduced FAD (7-10). C 2 can effectively stabilize the C4a-hydroperoxyflavin intermediate for minutes, and, at high concentration of HPA, a stable dead-end complex between C4a-hydroxyflavin and HPA is observed.Here, we present crystal structures of C 2 in the apoenzyme form and of its complexes with FMNH Ϫ (C 2 :FMNH Ϫ ) and HPA (C 2 :FMNH Ϫ :HPA). Structural analysis reported here provides a fram...

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“…These enzymes use external reducing agents (NAD(P)H; ascorb~te; H4-pterin) to provide electrons for the reduction of oxyge a to the level of hydrogen peroxide, which then is responsible fo" the insertion of oxygen into the substrate [25]. This fixation ol oxygen into the reaction products is an energy-consuming pr )cess.…”

Section: Discussionmentioning

confidence: 99%

“…F: tvin-containing oxygenases seem to be limited to those types ol oxidations that are possible with alkylperoxides or oxygen in the absence of a metal ion [25]. FAD is the most important c(,enzyme of those flavin-dependent monooxygenases [26].…”

Section: Discussionmentioning

confidence: 99%

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FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

1995

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dase was suggested to be associated with the stromal side of this membrane [16]. Recently, Zea epoxidation activity was attributed to isolated LHCsII [19,20].In this study we demonstrate that a further cofactor, flavin adenine dinucleotide (FAD), is involved in the NAD(P)H-and O2-dependent epoxidation of Zea. Presumable, one part of the protein-associated flavin moiety is lost during the isolation procedure of the thylakoid membranes and consequently the epoxidase activity is attenuated. Addition of FAD to the thylakoid suspensions restores the enzyme activity. A further indication for the importance of this flavin during the Zea epoxidation is the strong effect of DPI, an inhibitor of flavin-containing enzymes [21,22]. Materials and methods Plant materialSpinach plants (Spinacia oleracea L. cv. Butterfly) were cultivated in a growth chamber (10 h light, 23°C, 55% rel. humidity; 14 h dark, 15°C, 70% rel. humidity). The illumination period was started and terminated by a dawn and dusk period of 1 h. For illumination HQI-R 250 W bulbs (Osram) were used (140 pmol photons-m -2-s-%. All experiments were performed with 6-week-old leaves.

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Structure and mechanism of para‐hydroxybenzoate hydroxylase

Cited by 208 publications

References 0 publications

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

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Structure and mechanism of para‐hydroxybenzoate hydroxylase

Cited by 208 publications

References 0 publications

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

FAD is a further essential cofactor of the NAD(P)H and O2‐dependent zeaxanthin‐epoxidase

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FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase