4-hydroxybenzoate 3-monooxygenase

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4-hydroxybenzoate 3-monooxygenase
4-hydroxybenzoate 3-monooxygenase.png
Crystal structure of 4-hydroxybenzoate 3-monooxygenase
Identifiers
EC no. 1.14.13.2
CAS no. 9059-23-8
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The enzyme 4-hydroxybenzoate 3-monooxygenase, also commonly referred to as para-hydroxybenzoate hydroxylase (PHBH), is a flavoprotein belonging to the family of oxidoreductases. Specifically, it is a hydroxylase, and is one of the most studied enzymes and catalyzes reactions involved in soil detoxification, metabolism, and other biosynthetic processes. [1]

Contents

4-hydroxybenzoate 3-monooygenase catalyzes the regioselective hydroxylation of 4-hydroxybenzoate, giving 3,4-dihydroxybenzoate as the product. The mechanism consists of the following general steps: (1) reduction of the flavin, (2) reaction of the flavin with O2, producing C4a-hydroperoxyflavin, and (3) binding and activation of the substrate, leading to product formation and release. [2] Throughout the mechanism, the flavin changes between “open” and “closed” conformations, thus altering the substrate reaction environment. The open conformation allows solvent access to the active site; the enzyme adopts this conformation for substrate binding and product release. A closed conformation isolates the reaction from solvent, which helps to stabilize the reaction intermediates. [2]

The enzymatic conversion of 4-hydroxybenzoate to 3,4-dihydroxybenzoate P-hydroxybenzoate hydroxylase rxn scheme.jpg
The enzymatic conversion of 4-hydroxybenzoate to 3,4-dihydroxybenzoate

Structure

4-hydroxybenzoate 3-monooxygenase is a homodimer with a flavin bound to each monomer. The active site is composed of the flavin and amino acids on the monomer. The structure of this enzyme often serves as a model for structure-reactivity interdependence of other flavin-dependent hydroxylases. The active site limits potential substrates to substituted benzenes, namely 4-hydroxybenzoate (the native substrate), 2,4-dihydroxybenzoate, 4-mercaptobenzoate, and several halogenated aromatic compounds. [3]

Mechanism

The substrate is held in place by several non-covalent interactions with the protein scaffold. 4-hydroxybenzoate 3-monooxygenase bound substrate.png
The substrate is held in place by several non-covalent interactions with the protein scaffold.

The hydroxylase, 4-hydroxybenzoate 3-monooxygenase, proceeds through a catalytic process that begins with the entrance of NADPH and 4-hydroxybenzoate (the native substrate) into the active site of the enzyme. This results in formation of an enzyme-flavin-substrate-NADPH complex, after which the flavin cofactor, FAD, is reduced by NADPH. NADP+ is lost and O2 enters into the complex, followed by oxidation of the flavin to form a hydroperoxide, which acts as the hydroxide transfer reagent. It is important to note that while the group transferred is referred to as hydroxide it is formally an OH+ group. This hydroxide is transferred to the substrate from the hydroperoxide flavin, flavin-C4a-hydroperoxide, via an electrophilic aromatic substitution-type reaction. Finally, the product exits from the complex and the hydroxy-flavin is dehydrated, regenerating FAD and allowing the process to repeat. [2]

The substrate binds in the active site of the enzyme via non-covalent interactions with proximal amino acid side chains. Specifically, the hydroxyl groups of tyrosine 201 and 222, in addition to the hydroxyl group of serine 212, interact with the carboxylate and hydroxyl group on the substrate. This allows for proper orientation within the active site in order to achieve reactivity. [2]

Once the substrate is bound, the flavin shifts from an “open” to a “closed” conformation. This shields the active site and substrate from solvent, preventing the premature breakdown of the flavin hydroperoxide. Binding of both NADPH and substrate shifts the enzyme to an “out” conformation. This occurs through an intricate proton network within the enzyme that allows for deprotonation of the phenol and a subsequent dynamic shift of the enzyme. The “out” conformation aligns the isoalloxazine ring of the flavin so that it can be reduced rapidly by NADPH. Following the reduction, NADP+ is released from the enzyme. [4]

Reduction of the flavin generates a negatively charged species, FADH−, which is attracted to the positively charged active site. This attraction shifts the flavin back to the “closed” conformation, isolating it from the solvent environment. This isolation provides an optimal environment and position for O2 to hydroxylate the substrate. [4] The oxygen binds to FADH− via a single electron transfer, which is the rate-limiting step of the reaction. This forms an FAD radical and flavin hydroperoxide. Reaction between these generates C4a-peroxyflavin, which is quickly protonated to form flavin-C4a-hydroperoxide. [3] Tautomerization leads to the formation of 3,4-dihydoxybenzoate. The final step in the mechanism is dissociation of the product and water from FAD, causing the flavin to return to the open conformation. [1]

Related Research Articles

<span class="mw-page-title-main">Active site</span> Active region of an enzyme

In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

<span class="mw-page-title-main">Flavin adenine dinucleotide</span> Redox-active coenzyme

In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated with various proteins, which is involved with several enzymatic reactions in metabolism. A flavoprotein is a protein that contains a flavin group, which may be in the form of FAD or flavin mononucleotide (FMN). Many flavoproteins are known: components of the succinate dehydrogenase complex, α-ketoglutarate dehydrogenase, and a component of the pyruvate dehydrogenase complex.

4-Hydroxybenzoic acid, also known as p-hydroxybenzoic acid (PHBA), is a monohydroxybenzoic acid, a phenolic derivative of benzoic acid. It is a white crystalline solid that is slightly soluble in water and chloroform but more soluble in polar organic solvents such as alcohols and acetone. 4-Hydroxybenzoic acid is primarily known as the basis for the preparation of its esters, known as parabens, which are used as preservatives in cosmetics and some ophthalmic solutions. It is isomeric with 2-hydroxybenzoic acid, known as salicylic acid, a precursor to aspirin, and with 3-hydroxybenzoic acid.

Any enzyme system that includes cytochrome P450 protein or domain can be called a P450-containing system.

<span class="mw-page-title-main">Methane monooxygenase</span> Class of enzymes

Methane monooxygenase (MMO) is an enzyme capable of oxidizing the C-H bond in methane as well as other alkanes. Methane monooxygenase belongs to the class of oxidoreductase enzymes.

<span class="mw-page-title-main">Squalene monooxygenase</span> Mammalian protein found in Homo sapiens

Squalene monooxygenase is a eukaryotic enzyme that uses NADPH and diatomic oxygen to oxidize squalene to 2,3-oxidosqualene. Squalene epoxidase catalyzes the first oxygenation step in sterol biosynthesis and is thought to be one of the rate-limiting enzymes in this pathway. In humans, squalene epoxidase is encoded by the SQLE gene. Several eukaryote genomes lack a squalene monooxygenase encoding gene, but instead encode an alternative squalene epoxidase that performs the same task.

In enzymology, a 3-hydroxybenzoate 4-monooxygenase (EC 1.14.13.23) is an enzyme that catalyzes the chemical reaction

In enzymology, a 3-hydroxybenzoate 6-monooxygenase (EC 1.14.13.24) is an enzyme that catalyzes the chemical reaction

In enzymology, a 4-hydroxybenzoate 1-hydroxylase (EC 1.14.13.64) is an enzyme that catalyzes the chemical reaction

In enzymology, a 4-hydroxybenzoate 3-monooxygenase [NAD(P)H] (EC 1.14.13.33) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">4-Hydroxyphenylacetate 3-monooxygenase</span> Class of enzymes

4-hydroxyphenylacetate 3-monooxygenase (EC 1.14.14.9) is an enzyme that catalyzes the chemical reaction

In enzymology, an anthranilate 3-monooxygenase (deaminating) (EC 1.14.13.35) is an enzyme that catalyzes the chemical reaction

In enzymology, a benzoate 4-monooxygenase (EC 1.14.14.92, Formerly EC 1.14.13.12) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Kynurenine 3-monooxygenase</span> Enzyme

In enzymology, a kynurenine 3-monooxygenase (EC 1.14.13.9) is an enzyme that catalyzes the chemical reaction

In enzymology, a salicylate 1-monooxygenase (EC 1.14.13.1) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Monooxygenase</span>

Monooxygenases are enzymes that incorporate one hydroxyl group (−OH) into substrates in many metabolic pathways. In this reaction, the two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation of NAD(P)H. One important subset of the monooxygenases, the cytochrome P450 omega hydroxylases, is used by cells to metabolize arachidonic acid (i.e. eicosatetraenoic acid) to the cell signaling molecules, 20-hydroxyeicosatetraenoic acid or to reduce or totally inactivate the activate signaling molecules for example by hydroxylating leukotriene B4 to 20-hydroxy-leukotriene B5, 5-hydroxyeicosatetraenoic acid to 5,20-dihydroxyeicosatetraenoic acid, 5-oxo-eicosatetraenoic acid to 5-oxo-20-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid to 12,20-dihydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acids to 20-hydroxy-epoxyeicosatrienoic acids.

Cyclohexanone monooxygenase (EC 1.14.13.22, cyclohexanone 1,2-monooxygenase, cyclohexanone oxygenase, cyclohexanone:NADPH:oxygen oxidoreductase (6-hydroxylating, 1,2-lactonizing)) is an enzyme with systematic name cyclohexanone,NADPH:oxygen oxidoreductase (lactone-forming). This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Tryptophan 7-halogenase</span>

Tryptophan 7-halogenase (EC 1.14.19.9, PrnA, RebH) is an enzyme with systematic name L-tryptophan:FADH2 oxidoreductase (7-halogenating). This enzyme catalyses the following chemical reaction:

<span class="mw-page-title-main">Flavin-containing monooxygenase</span> Class of enzymes

The flavin-containing monooxygenase (FMO) protein family specializes in the oxidation of xeno-substrates in order to facilitate the excretion of these compounds from living organisms. These enzymes can oxidize a wide array of heteroatoms, particularly soft nucleophiles, such as amines, sulfides, and phosphites. This reaction requires an oxygen, an NADPH cofactor, and an FAD prosthetic group. FMOs share several structural features, such as a NADPH binding domain, FAD binding domain, and a conserved arginine residue present in the active site. Recently, FMO enzymes have received a great deal of attention from the pharmaceutical industry both as a drug target for various diseases and as a means to metabolize pro-drug compounds into active pharmaceuticals. These monooxygenases are often misclassified because they share activity profiles similar to those of cytochrome P450 (CYP450), which is the major contributor to oxidative xenobiotic metabolism. However, a key difference between the two enzymes lies in how they proceed to oxidize their respective substrates; CYP enzymes make use of an oxygenated heme prosthetic group, while the FMO family utilizes FAD to oxidize its substrates.

<span class="mw-page-title-main">L-ornithine N5 monooxygenase</span> Enzyme

L-ornithine N5 monooxygenase (EC 1.14.13.195 or EC 1.14.13.196) is an enzyme which catalyzes one of the following chemical reactions:

L-ornithine + NADPH + O2 N(5)-hydroxy-L-ornithine + NADP+ + H2O L-ornithine + NAD(P)H + O2 N(5)-hydroxy-L-ornithine + NAD(P)+ + H2O

References

  1. 1 2 Entsch B, Ballou DP, Begley TP (2007-01-01). Wiley Encyclopedia of Chemical Biology. John Wiley & Sons, Inc. doi:10.1002/9780470048672.wecb672. ISBN   9780470048672.
  2. 1 2 3 4 Gatti DL, Palfey BA, Lah MS, Entsch B, Massey V, Ballou DP, Ludwig ML (October 1994). "The mobile flavin of 4-OH benzoate hydroxylase". Science. 266 (5182): 110–4. Bibcode:1994Sci...266..110G. doi:10.1126/science.7939628. PMID   7939628.
  3. 1 2 Montersino S, Tischler D, Gassner GT, van Berkel WJ (September 2011). "Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases". Advanced Synthesis & Catalysis. 353 (13): 2301–2319. doi:10.1002/adsc.201100384.
  4. 1 2 Ballou DP, Entsch B, Cole LJ (December 2005). "Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases". Biochemical and Biophysical Research Communications. 338 (1): 590–8. doi:10.1016/j.bbrc.2005.09.081. PMID   16236251.

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