Kinetics of a Two-Component <i>p</i>-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase

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Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

Section: Discussioncontrasting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

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“…FMN-dependent systems are involved in the synthesis of antibiotics such as actinorhodin in Streptomyces coelicolor (23), the bacterial degradation of polyaminocarboxylates such as nitrilotriacetic acid (24), and the desulfurization of fossil fuels by rhodococci (25). 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).…”

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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...

“…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).…”

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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. X-ray structures of the oxygenase components from A. baumannii at 2.3-2.8 Å resolution (23) and Thermus thermophilus HB8 at 1.3-2.0 Å resolution (24,25) and the reductase component from Sulfolobus tokodaii (26) at 1.7-2.3 Å resolution have been solved.…”

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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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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 *