The Reductase of p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii Requires p-Hydroxyphenylacetate for Effective Catalysis

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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 *

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

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“…FMN was prepared by conversion of FAD to FMN with snake venom from Crotalus adamanteus (19). DCPIP and menadione were from Sigma.…”

Section: Materials-nadmentioning

confidence: 99%

“…Recently, it was proposed that the activity of the monooxygenase component in the A. baumannii system was limited by the transfer of FMN red from the reductase, HPAH-C 1 , to the monooxygenase, HPAH-C 2 (27). Furthermore, HPA, the monooxygenase substrate, was shown to function as an effector of HPAH-C 1 for both FMN reduction and FMN red release (19,27). Thus, as in the case of the ActVAActVB system, HPAH-C 1 , the reductase component, is likely to regulate the overall monooxygenase activity.…”

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confidence: 99%

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

Valton

Mathevon

Fontecave

et al. 2008

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The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. , 7), in the utilization of sulfur from aliphatic sulfonates in E. coli (8), and in the desulfurization of fossil fuels by Rhodococcus species (9), as well as the hydroxylation of 4-hydroxyphenylacetate in Acinetobacter baumannii (10). We have been investigating the mechanism of the FMN-dependent twocomponent enzyme system, ActVA-ActVB, which participates in the last steps of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor (11-13) (Scheme 1).The first enzyme of the two-component flavin-dependent oxygenases is a NAD(P)H:flavin oxidoreductase that catalyzes the reduction of free oxidized flavins (FAD and/or FMN) by the reduced pyridine nucleotides, NADPH or NADH (3,[14][15][16]. The second enzyme is an oxygenase that binds the resulting free reduced flavin and promotes its reaction with O 2 to hydroxylate the substrate (2,3,(5)(6)(7)(8)(9)(10)17). Therefore, in contrast to the single-component flavin hydroxylases, the two-component sys-* This work was supported by National Institutes of Health Grant GM64711 (to D. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed.

“…pHPAH (EC 1.14.13.3), a well characterized FMN-utilizing enzyme from Acinetobacter baumanii, catalyzes the hydroxylation of p-hydroxyphenyl acetate (HPA) to 3,4-dihydroxyphenyl acetate (PDB entry 2JBT) (26). The reductase component, C 1 , requires HPA for effective flavin reduction (27), and the oxygenase component, C 2 , binds FMNH 2 prior to HPA (28). HsaAB has been predicted to be a TC-FDM based on the ϳ32% amino acid sequence identity that it shares with C 2 and C 1 of pHPAH.…”

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confidence: 99%

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Dresen

Lin

D’Angelo

et al. 2010

100

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-). However, 3,9-DHSA was a poorer substrate than 3-hy-). In the presence of 3-HSA the K mapp for O 2 was 100 ؎ 10 M. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val 367 -Val 394 ) could adopt two conformations differing by a rigid body rotation of 25°around Arg 366 . This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids. Mycobacterium tuberculosis (Mtb),6 the most devastating infectious agent of mortality worldwide (1), remains a primary public health threat due to synergy with the human immunodeficiency virus and the emergence of extensively drug-resistant strains. Mtb has the unusual ability to survive and replicate in the hostile environment of the macrophage (2, 3) utilizing largely unknown mechanisms. Genes essential to intracellular survival have been identified using genome-wide mutagenesis (4). A cluster of such genes was subsequently predicted to specify cholesterol catabolism (5). Targeted gene deletion studies subsequently indicated that cholesterol catabolism occurs throughout infection, contributing to the virulence of Mtb (6 -8). Although the exact role of cholesterol in infection remains unclear, the gene deletion studies are consistent with the high concentrations of cholesterol found within caseating granulomas and the observed congregation of bacteria around cholesterol foci (9).The cholesterol catabolic pathway of Mtb is very similar to that of other pathogenic and environmental actinomycetes (10), as initially predicted in Rhodococcus jostii RHA1 (5). Sidechain degradation is initiate...