Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase

Entsch, Barrie; Cole, Lindsay J.; Ballou, David P.

doi:10.1016/j.abb.2004.09.029

Cited by 132 publications

(188 citation statements)

References 47 publications

Supporting

Mentioning

184

Contrasting

Order By: Relevance

“…Each of these three homologues catalyze flavin-mediated hydroxylations on the ortho position of electron-rich phenol derivatives (SI Fig. 8) (13,15,16).…”

Section: Resultsmentioning

confidence: 99%

Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

Ryan

Howard‐Jones²,

Hamill³

et al. 2007

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

102

View full text Add to dashboard Cite

The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product.flavin enzymes ͉ x-ray crystallography ͉ indolocarbazoles ͉ antitumor R ebeccamycin (1, Fig. 1a) is a natural product isolated from Lechevalieria aerocolonigenes (1) and is a prototype for a class of compounds that bind to DNA-topoisomerase I complexes (2), preventing the replication of DNA and thereby acting as antitumor compounds (3). Rebeccamycin is synthesized by the action of eight enzymes, with the overall conversion of Ltryptophan, chloride, molecular oxygen, glucose, and a methyl group to the glycosylated indolocarbazole rebeccamycin (1) (4-9). One step in this pathway is the conversion of chlorinated chromopyrrolic acid (2) to the rebeccamycin aglycone (3); this reaction involves an eight-electron oxidation, and it is carried out by the enzyme pair RebP and RebC (10). The mechanism of this reaction is not established (8).RebP is annotated as a cytochrome P450, and it is functionally equivalent to its homologue StaP from the staurosporine biosynthetic pathway (10), which has been used in place of RebP in initial biochemical investigations. Additionally, all biochemical work on StaP and RebC has used nonchlorinated substrates because they are well tolerated by both enzymes (8). One of the most remarkable features of StaP is that when it is incubated alone with its nonchlorinated substrate, chromopyrrolic acid (4), as well as with an electron source (provided by ferredoxin, flavodoxin NADP ϩ -reductase, and NAD(P)H), StaP is capable of generating a number of products, including arcyriaflavin A (5), 7-hydroxy-K252c (6), and K252c (7) at a 1:7:1 ratio (8) (Fig. 1b). Inclusion of RebC, an FAD-containing protein, in the reaction mixture results in near-exclusive production of arcyriaflavin A (5) and acceleration of turnover (8). One possibility to account for this phenomenon is that RebC can direct the outcome of the reaction by mediating the catalytic activity of StaP through a protein-protein interaction rather than acting catalytically itself. Although no stable interaction was found between StaP and RebC through pull-down assays (8), a transient complex cannot be ruled out. Another possibility...

show abstract

“…Each of these three homologues catalyze flavin-mediated hydroxylations on the ortho position of electron-rich phenol derivatives (SI Fig. 8) (13,15,16).…”

Section: Resultsmentioning

confidence: 99%

Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

Ryan

Howard‐Jones²,

Hamill³

et al. 2007

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

102

View full text Add to dashboard Cite

show abstract

“…An electrophilic aromatic substitution mechanism has been shown to occur in p-hydroxybenzoate hydroxylase, a singlecomponent flavin-dependent monooxygenase that catalyses a reaction similar to that carried out by C 2 (the hydroxylation of 4-hydroxybenzoate) (22). Inspection of the three-dimensional structures shows that the geometry of substrate-binding site is very different in the two enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…The former case is observed in Baeyer-Villiger monooxygenases (such as cyclohexanone monooxygenase) (19) or bacterial luciferase (20) in which the flavin C4a-peroxide acts as a nucleophile attacking the substrate. On the contrary, a flavin C4a-hydroperoxide has been well documented in flavin-containing monooxygenases (21) and aromatic hydroxylases (22)(23)(24), in which the terminal oxygen of hydroperoxyflavin acts as an electrophile in an aromatic substitution reaction. The three-dimensional structure suggests that an electrophilic aromatic substitution mechanism is likely to occur also in the C 2 reaction.…”

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

View full text Add to dashboard Cite

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...

show abstract

“…For the PHBH reaction, the hydroxylation rate constant increases upon an increase in pH with a pK a of 7.1 (13). This pK a value corresponds to the pK a of His-72, which is located on the protein surface and controls the H-bond network that facilitates removal of the phenolic proton of pOHB (9,13,14). For two-component flavin-dependent monooxygenases, the mode of oxygen atom transfer in hydroxylation of aromatic compounds has never been explored.…”

mentioning

confidence: 99%

“…The best understood oxygenation reaction is the hydroxylation of aromatic compounds catalyzed by a single-component flavoenzyme, p-hydroxybenzoate (pOHB) 5 hydroxylase (PHBH) (4,6,9). Studies of PHBH and 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) monooxygenase (MHPCO) reactions showed that hydroxylation occurs via an electrophilic aromatic hydroxylation mechanism in which an aromatic substrate acts as a nucleophile and a terminal -OH group of C4a-hydroperoxy flavin acts as an electrophile (9 -12).…”

mentioning

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

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 *

show abstract

Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase

Cited by 132 publications

References 47 publications

Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC

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

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

Contact Info

Product

Resources

About