Cloning and expression of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii: evidence of the divergence of enzymes in the class of two-protein component aromatic hydroxylases

Thotsaporn, Kittisak; Sucharitakul, Jeerus; Wongratana, Janewit; Suadee, Chutintorn; Chaiyen, Pimchai

doi:10.1016/j.bbaexp.2004.08.003

Cited by 69 publications

(103 citation statements)

References 30 publications

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“…4). With such an arrangement, the additional AMP moiety of FAD would easily hang out in the solvent or rest on the tetramer surface; this observation explains why both FMNH Ϫ and FADH Ϫ work equally well in the hydroxylation reaction (7,9).…”

Section: Resultsmentioning

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.

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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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Section: Resultsmentioning

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.

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133

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“…P. entomophila shares several gene clusters with P. putida 27 that are involved in the degradation of various classes of aromatic compounds including benzoate and quinate, 4-hydroxybenzoate, phenylacetaldehyde and phenylalkanoate as well as phenylalanine and tyrosine. The P. entomophila genome contains two additional catabolic gene clusters present in the genome of P. aeruginosa PAO1 that encode determinants for the degradation of 3-hydroxybenzoate through gentisate 28 and for the meta-cleavage of homoprotocatechuate 29,30 .…”

Section: Metabolism Transport and Regulationmentioning

confidence: 99%

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

et al. 2006

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Pseudomonas entomophila is an entomopathogenic bacterium that, upon ingestion, kills Drosophila melanogaster as well as insects from different orders. The complete sequence of the 5.9-Mb genome was determined and compared to the sequenced genomes of four Pseudomonas species. P. entomophila possesses most of the catabolic genes of the closely related strain P. putida KT2440, revealing its metabolically versatile properties and its soil lifestyle. Several features that probably contribute to its entomopathogenic properties were disclosed. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather relies on a number of potential virulence factors such as insecticidal toxins, proteases, putative hemolysins, hydrogen cyanide and novel secondary metabolites to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that regulates most of the potential virulence factors identified.

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“…High purity FMN was produced by converting FAD to FMN using snake venom from Crotalus adamanteus (28) according to the protocol described in Sucharitakul et al (22). C 1 , wild-type C 2 , and C 2 variants were expressed and purified as previously described (15)(16)(17). The concentrations of the following compounds were determined using the known extinction coefficients at pH 7. enzyme solution was reduced with an equal reducing equivalent of sodium dithionite (a solution of 5 mg/ml in 100 mM potassium phosphate buffer pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

“…The quenched samples were collected from the sample loop, and the enzyme was removed by ultrafiltration with a Microcon unit (Amicon YM-10). The filtrates were analyzed for the amount of DHPA produced from the reaction using an HPLC method, as previously described (16,17).…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme from Acinetobacter baumannii is composed of a reductase component (C 1 ) and an oxygenase component (C 2 ), and it catalyzes the hydroxylation of HPA to 3,4-dihydroxyphenylacetate (DHPA) (16,17). Kinetic studies of HPAHs from Pseudomonas putida (18), Pseudomonas aeruginosa (19), Escherichia coli W (20), and A. baumannii (8,15,21,22) have been reported.…”

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Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Tongsook

Sucharitakul

Thotsaporn

et al. 2011

Journal of Biological Chemistry

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plex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ϳ70% over a pH range of 6 -10, whereas product formation for S146C decreased from ϳ65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the His ␦؉ ⅐HPA ␦؊ complex needed for hydroxylation.Incorporation of single oxygen atoms into organic compounds (monooxygenation) by hydroxylation or epoxidation is an important biological process for aerobic organisms. The monooxygenation reaction is generally catalyzed by cytochrome P450, metalloenzymes, pterin-dependent and flavindependent monooxygenases (1). Flavin-dependent monooxygenases are involved in a wide variety of biological processes (2, 3). These enzymes catalyze the monooxygenation of many aromatic and aliphatic compounds (3).Based on the number of proteins required for catalysis, flavin-dependent monooxygenases can be classified into singleprotein component and two-protein component types (2-5). Besides an organic substrate, both types of enzymes require NAD(P)H and oxygen as co-substrates. The initial part of the reaction is the reduction of an enzyme-bound flavin by NAD(P)H to generate reduced flavin followed by the reaction of reduced flavin with oxygen to form the C4a-(hydro)peroxy flavin, a key intermediate that is required for oxygenation of an organic substrate. Oxygenation occurs via an oxygen atom transfer from C4a-hydroperoxy flavin to an organic substrate, resulting in the formation of a C4a-hydroxy flavin intermediate and an oxygenated product. The C4a-hydroxy flavin dehydrates at the last step of the reaction to form the final species, oxidized flavin (2-4, 6). All steps for the reactions of singleprotein component flavin-dependent monooxygenases occur within the same polypeptide, whereas for the two-protein component type the flavin reduction occurs on a reductase component and the oxygenation occurs on an oxygenase component (4 -8). The mechanism by which the reduced flavin is transferred involves simple diffusion or protein-protein contacts (5,8). Although single-component flavin-dependent monooxygenases have been studied for more than 40 years, two-component flavin-dependent monooxygenases have only received significant attention during the past decade after recent discoveries of their involvement in a wide variety of reactions (2, 5).The mechanism by which the oxygen atom transfer occurs in flavin-dependent monooxygenases is well understood for only a few enzymatic systems. The best understood oxygenation reaction is the hydroxylation of aromatic compounds catalyzed *

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Cloning and expression of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii: evidence of the divergence of enzymes in the class of two-protein component aromatic hydroxylases

Cited by 69 publications

References 30 publications

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

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

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

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