Publications

• 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 K., Sucharitakul J., Wongratana J., Suadee C., Chaiyen P.

  The genes encoding for the reductase and oxygenase components of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in an E. coli system. The recombinant enzymes were purified and shown to have the same catalytic properties as the native enzyme. Sequence an ... >> More

  The genes encoding for the reductase and oxygenase components of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in an E. coli system. The recombinant enzymes were purified and shown to have the same catalytic properties as the native enzyme. Sequence analysis and biochemical studies indicate that the enzyme represents a novel prototype of enzyme in the two-protein component class of aromatic hydroxylases. The C2 component shows little similarity to other oxygenases in the same class, correlating with its uniquely broad flavin specificity. Analysis of the C1 reductase sequence indicates that the binding sites of flavin and NADH mainly reside in the N-terminal half while the C-terminal half may be responsible for HPA-stimulation of NADH oxidation. << Less

  Biochim. Biophys. Acta 1680:60-66(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Alfieri A., Fersini F., Ruangchan N., Prongjit M., Chaiyen P., Mattevi A.

  p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket ... >> More

  p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) 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 C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-A radius and a 29-A3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate. << Less

  Proc. Natl. Acad. Sci. U.S.A. 104:1177-1182(2007) [PubMed] [EuropePMC]

• Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii.

  Sucharitakul J., Chaiyen P., Entsch B., Ballou D.P.

  p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C1) and an oxygenase that uses FMNH-(C2). We report detaile ... >> More

  p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C1) and an oxygenase that uses FMNH-(C2). We report detailed transient kinetics studies at 4 degrees C of the reaction mechanism of C2.C2 binds rapidly and tightly to reduced FMN (Kd, 1.2 +/- 0.2 microm), but less tightly to oxidized FMN (Kd, 250 +/-50 microm). The complex of C -FMNH-2 reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 +/-0.1 x 10(6) m(-1) s(-1), whereas the C -FMNH-2 -HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 +/-0.2 x 10(4) m(-1) s(-1)). The kinetic mechanism of C2 was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C -FMNH-2 complex, but the preferred path was oxygen reacting with C -FMNH-2 to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20 s(-1) to form the C2-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mm), HPA formed a dead end complex with the C2-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH-was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH-. Thus, C2 has the unusual ability to use both common flavin cofactors in catalysis. << Less

  J. Biol. Chem. 281:17044-17053(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel two-protein component flavoprotein hydroxylase.

  Chaiyen P., Suadee C., Wilairat P.

  p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation ... >> More

  p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C2) is the hydroxylase component that hydroxylates HPA in the presence of C1. C2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)-substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH2, FADH2, or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases. << Less

  Eur. J. Biochem. 268:5550-5561(2001) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.