User:Jgmurphy102/sandbox
4-hydroxybenzoate 3-monooxygenase
[edit]| 4-hydroxybenzoate 3-monooxygenase | |||||||||
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| Identifiers | |||||||||
| EC no. | 1.14.13.2 | ||||||||
| CAS no. | 9059-23-8 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
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4-hydroxybenzoate 3-monooxygenase, also commonly referred to as para-hydroxybenzoate hydroxylase (PHBH), is a flavoprotein belonging to the family of oxidoreductases. It is one of the most studied enzymes and catalyzes reactions involved in soil detoxification, metabolism, and other biosynthetic processes.[1]
4-hydroxybenzoate 3-monooygenase catalyzes the regioselective hydroxylation of 4-hydroxybenzoate, giving the product 3,4-dihydroxybenzoate. 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
Structure
[edit]4-hydroxybenzoate 3-monooxygenase is a homodimer with a flavin bound to each monomer. The active site is comprised 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
[edit]
The hydroxylase, 4-hydroxy 3-monooxygenase, proceeds through a catalytic process that involves: (1) NADPH and p-hydroxybenzoate enter the active site of the enzyme, resulting in formation of an enzyme-flavin-substrate-NADPH complex, after which NADPH reduces the flavin; (2) loss of NADP+ and entrance of O2 into the complex, followed by oxidation of the flavin to form a hydroperoxide, which acts as the hydroxide transfer reagent; (3) the hydroxide is transferred to the substrate from the hydroperoxide flavin, flavin-C4a-hydroperoxide, via an electrophilic aromatic substitution-type reaction; (4) the substrate leaves the complex, and the hydroxy-flavin undergoes a dehydration reaction to regenerate FAD, allowing for 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]
References
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Comments for Peer Review
[edit]Hey Guys!
I think it's important for you guys to reference the website where you are getting the names from. Speaking of references, wikipedia actually makes it remarkably easy for you to include citations in your articles (look at mine! I'm lazy and it came out that good). I can show Bates sometime if you need to see it. Also, you say 'para-' in the second paragraph but you should shorten it to p- since that's what you use the paragraph before. You should consider including the catalytic cycle with pictures, as a lot of these mechanistic steps are difficult to imagine (I can show Bates how to upload pictures too).
More to come soon <3
Arvin:
Hello! I noticed there isn't too much information readily available from a google search about the structure of these enzymes. Perhaps include a section dedicated specifically to important amino acid residues in the active site or general scaffolds/tertiary structure that is retained throughout these systems.
~~~~
Hey guys!
It's ya boi again! I think Arvin is completely right, you should absolutely include more detail about the structure of the enzymes, and see if there's any literature that connects the structure to the catalysis like we've seen in class. I think the part of the article where you discuss the tyrosine and serine residues involved in binding the substrate would be a great place to include either a pymol picture or something in chemdraw, if you decide to go that route instead of showing the catalytic cycle like I suggested before.
This is Jerry,
-Arvin and Harrison make great points. I would like to mention again the pymol image comment for the mechanism. I would also say maybe expanding the article in more directions such as functional relevance; what's interesting about it.
-Also, should we delete the nomenclature section or just condense it to a sentence somehow?
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A Julia comment:
We have to be consistent with in/out/open/closed description of the flavin. If I'm wrong and these aren't all the same, we need to clarify the meaning of each.
- ^ a b Entsch, Barrie; Ballou, David P.; Begley, Tadhg P. (2007-01-01). Wiley Encyclopedia of Chemical Biology. John Wiley & Sons, Inc. doi:10.1002/9780470048672.wecb672. ISBN 9780470048672.
- ^ a b c d Gatti, D. L.; Palfey, B. A.; Lah, M. S.; Entsch, B.; Massey, V.; Ballou, D. P.; Ludwig, M. L. (1994-10-07). "The mobile flavin of 4-OH benzoate hydroxylase". Science. 266 (5182): 110–114. doi:10.1126/science.7939628. ISSN 0036-8075. PMID 7939628.
- ^ a b Montersino, Stefania; Tischler, Dirk; Gassner, George T.; van Berkel, Willem J. H. (2011-09-01). "Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases". Advanced Synthesis & Catalysis. 353 (13): 2301–2319. doi:10.1002/adsc.201100384. ISSN 1615-4169.
- ^ a b Ballou, David P.; Entsch, Barrie; Cole, Lindsay J. (2005-12-09). "Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases". Biochemical and Biophysical Research Communications. Celebrating 50 Years of Oxygenases. 338 (1): 590–598. doi:10.1016/j.bbrc.2005.09.081.