4-hydroxyphenylacetate 3-monooxygenase
PyMOL-generated cartoon of a Thermus thermophilus hpaB monomer complexed to FAD and 4-hydroxyphenylacetate. Color-coding distinguishes the enzyme's three primary structural domains.[1]
Identifiers
EC no.	1.14.14.9
CAS no.	37256-71-6
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / QuickGO
Search PMC articles PubMed articles NCBI proteins

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

4-hydroxyphenylacetate + FADH₂ + O₂ ${\displaystyle \rightleftharpoons }$ 3,4-dihydroxyphenylacetate + FAD + H₂O

This reaction is the first step in a pathway found in enteric bacteria such as Escherichia coli and soil bacteria such as Pseudomonas putida which degrades 4-hydroxyphenylacetate (4-HPA), allowing these bacteria to use 4-HPA and other aromatic compounds found in mammalian digestive tracts or in soil as a carbon source.^[2] While most known flavin monooxygenases use NADH or NADPH as substrates (and use the flavins FAD or FMN as prosthetic groups ^[3]), this enzyme is part of a two-component system, in which a flavin oxidoreductase partner (EC 1.5.1.37) regenerates FADH₂ by oxidizing NADH to NAD⁺. hpaB and hpaC, the 4-HPA oxygenase and reductase partner proteins (respectively) of E. coli strain W, were the first two-component flavin monoxygenase system identified.^[4] While known examples of this enzyme share a common catalytic mechanism and likely evolutionary origin, they differ with respect to regulation and ability to substitute FMNH₂ for FADH₂ as a substrate.

This enzyme is a tetramer which forms as a dimer of dimers.^[1] Sequence alignments of the Thermus thermophilus and E. coli hpaB enzymes show structural similarity to each other and to the oxygenase components of other bacterial two-component monooxygenases for compounds such as phenol and chlorophenol.^[1] Each monomer of this protein consists of an N-terminal alpha-helical domain, a middle beta barrel domain, and a C-terminal "tail" helix. FADH₂ is bound by a groove between these domains; this binding event causes a loop in the middle domain to change position, preforming a binding site for 4-HPA. At this point, multiple residues act to stabilize catalytic intermediates by hydrogen-bonding to the peroxide bound to FAD, as well as to the hydroxyl group of 4-HPA's phenol moiety (stabilizing its dienone transition state - see Mechanism). The loop which moves to form the 4-HPA binding site when FADH₂ binds provides catalytic specificity by hydrogen-bonding to 4-HPA's carboxylic acid moiety.^[1]

While many flavin-dependent monooxygenases retain FMN or FAD in their active sites throughout their catalytic cycle,^[3] this enzyme binds FADH₂ at the start of its catalytic cycle and releases FAD at the end. After FADH₂ binds, dioxygen attacks it at the C4a carbon, leading to a C4a-hydroperoxyflavin intermediate. 4-HPA then binds, and a conformational change sequesters the enzyme's active site from solution, preventing the oxygen from escaping as toxic hydrogen peroxide. 4-HPA is then hydroxylated via a dienone intermediate and released as 3,4-DHPA; with the peroxide bond broken, C4a-hydroxyflavin releases the remaining oxygen atom by eliminating water and is released from the enzyme as FAD.^[1]

To cope with the wide variety of phenolic compounds found in nature, bacterial pathways for degradation of aromatic compounds generally begin by channeling these diverse substrates towards a few common intermediates, which are then further degraded.^[5] Pathways for catabolizing aromatic compounds generally begin by adding two hydroxyl groups to the benzene ring, most often on adjacent carbons. This product then undergoes ring cleavage to produce a linear molecule which is ultimately degraded to intermediates of central metabolism, such as succinate, pyruvate, and carbon dioxide. In the case of 4-hydroxyphenylacetate, which only needs one hydroxyl group to be added, a monooxygenase is required.

Among common E. coli laboratory strains, the 4-HPA degradation pathway is not found in the commonly used K-12 strain, but it does appear in strains B, C, and W (the strain in which most research into the pathway has been done). Only strains containing this pathway are able to survive by metabolizing 4-HPA as the sole carbon source, showing that this pathway is required for catabolism of this compound, and likely for similar phenolic compounds as well.^[6]

In addition to the exclusively FADH₂-dependent monooxygenase found in species such as E. coli and T. thermophilus, a version of the enzyme which can use either reduced FAD or reduced FMN has been described in Acinetobacter baumannii.^[7] Although these enzymes catalyze similar reactions and share a similar acyl-CoA dehydrogenase fold, the FMN-dependent enzyme is allosterically regulated by 4-HPA while the FADH₂-dependent enzyme is likely regulated by a simpler kinetic mechanism, in which low concentrations of 4-HPA lead the enzyme to bind and sequester FADH₂ as part of the normal catalytic cycle, causing the free concentration of FADH₂ to drop until the flavin oxidoreductase's reaction is indirectly inhibited as well.^[8] Along with other differences in regulation and substrate binding despite a shared fundamental catalytic mechanism,^[9] this has led to speculation that these enzymes collectively represent an example of convergent evolution.^[3]

In addition to 4-HPA, the enzyme has been reported to have activity on a number of other phenolic substrates.^[4] Metabolic engineers have demonstrated that microbes expressing the E. coli enzyme can catalyze reactions such as hydroxylation of tyrosine to L-dopa^[10] and hydroxylation of the pharmacologically interesting phenylpropanoids umbelliferone and resveratrol.^[11] However, these approaches are not currently used commercially.