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Literature summary extracted from

  • Westphal, A.H.; Tischler, D.; van Berkel, W.J.H.
    Natural diversity of FAD-dependent 4-hydroxybenzoate hydroxylases (2021), Arch. Biochem. Biophys., 702, 108820 .
    View publication on PubMed

Application

EC Number Application Comment Organism
1.14.13.24 analysis 3-hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.14.13.24) from Rhodococcus jostii RHA1 is used as a lipid-containing reference protein for enzyme 4-hydroxybenzoate 1-hydroxylase, 4HB1H (EC 1.14.13.64). Models for Cp_4HB1H from Candida parapsilosis strain CBS604, Fo_4HB1H-like from Fusarium oxysporum, and VibMO1 from Boreostereum vibrans (UniProt ID A0A167KUL3) are generated using Rhodococcus jostii Rj_3HB6H (PDB ID 4bk1) and Pseudomonas putida Pp_SALH (PDB ID 5evy) as template structures, overview Rhodococcus jostii

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.2 gene phhA, sequence comparisons and phylogenetic analysis and tree Aspergillus niger
1.14.13.23 gene mobA, phylogenetic analysis Comamonas testosteroni
1.14.13.64 gene mnx1, sequence comparisons and phylogenetic analysis and tree, recombinant expression of C-terminally His-tagged enzyme in Escherichia coli strain TOP10 Candida parapsilosis

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.23 V257A a directed evolution study reveals that 3HB4H from Comamonas testosteroni strain GZ39 is considerably active with 4-hydroxybenzoate and that this activity increases in the V257A variant. The same variant slowly converts phenol to catechol, an activity not observed with the wild-type enzyme Comamonas testosteroni

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.64 0.01
-
4-hydroxybenzoate recombinant enzyme, pH 7.6, 25°C Candida parapsilosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Aspergillus niger
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 Aspergillus niger N402
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.23 3-hydroxybenzoate + NADPH + H+ + O2 Comamonas testosteroni
-
3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.24 3-hydroxybenzoate + NADH + H+ + O2 Rhodococcus jostii
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
?
1.14.13.64 4-hydroxybenzoate + NADH + 2 H+ + O2 Candida parapsilosis
-
hydroquinone + NAD+ + H2O + CO2
-
?
1.14.13.64 4-hydroxybenzoate + NADH + 2 H+ + O2 Candida parapsilosis CBS604
-
hydroquinone + NAD+ + H2O + CO2
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.2 Aspergillus niger
-
-
-
1.14.13.2 Aspergillus niger N402
-
-
-
1.14.13.23 Comamonas testosteroni Q6SSJ6
-
-
1.14.13.24 Rhodococcus jostii
-
-
-
1.14.13.64 Candida parapsilosis
-
-
-
1.14.13.64 Candida parapsilosis CBS604
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
1.14.13.24 lipoprotein the phospholipid has been proposed to be important for orienting the 3-HB substrate of 3HB6H for regioselective hydroxylation at C6 Rhodococcus jostii
1.14.13.64 additional information 4HB1H does not contain a lipid cofactor Candida parapsilosis

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.64 recombinant His-tagged enzyme from Escherichia coli strain TOP10 by nickel affinity chromatography, dialysis, ion exchange chromatography, and gel filtration Candida parapsilosis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2 = 3,4-dihydroxybenzoate + NADP+ + H2O the catalytic mechanism of PHBH from Pseudomonas species includes the reductive half-reaction, in which 4-hydroxybenzoate (4-HB) and some substrate analogues can act as effectors by stimulating the two-electron reduction of the FAD cofactor by NAD(P)H. In the oxidative half-reaction, the enzyme activates molecular oxygen through formation of a transiently stable flavin C4a-hydroperoxide and subsequently regioselectively incorporates one oxygen atom into the substrate. Based on the structural and kinetic properties of wild-type enzyme and a range of protein variants, the catalytic cycle of PHBH has been proposed to involve flavin movements in and out of the active site and opening and closure of a substrate transport channel. The C3 atom of the aromatic ring of the substrate is in close vicinity of the C4a atom of the isoalloxazine ring of FAD, which occupies the conformation. This active site configuration allows the attack of the flavin C4a-hydroperoxide oxygenation species onto the C3 atom of the substrate. The electrophilic ortho-hydroxylation reaction is stimulated by deprotonation of the phenolic moiety of 4-hydroxybenzoate, as facilitated by a water involved proton relay network that connects Tyr201 via Tyr385 and His72 with the protein surface Aspergillus niger
1.14.13.64 4-hydroxybenzoate + NAD(P)H + H+ + O2 = hydroquinone + NAD(P)+ + H2O + CO2 the conversion of 4-HB involves an ipso-attack of the electrophilic flavin C4a-hydroperoxide at the C1-atom of the activated substrate, resulting in a tetrahedral benzoquinone species and subsequent release of the carboxyl side chain. It cannot be excluded that His230 activates 4-HB in 4HB1H for ipso-attack by the flavin C4a-hydroperoxide. Furthermore, it can also not be ruled out that the substrate of 4HB1H binds in a flipped orientation with the 4-hydroxyl moiety pointing towards the side chains of Tyr116 and His118 Candida parapsilosis

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.64 14
-
purified recombinant enzyme, pH 7.6, 25°C, substrates 4-hydroxybenzoate and NADH Candida parapsilosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Aspergillus niger 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 4-hydroxybenzoate + NADPH + H+ + O2
-
Aspergillus niger N402 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.2 additional information substrate binding analysis and structure, each subunit of the dimeric enzyme contains a split substrate-binding domain, overview Aspergillus niger ?
-
-
1.14.13.2 additional information substrate binding analysis and structure, each subunit of the dimeric enzyme contains a split substrate-binding domain, overview Aspergillus niger N402 ?
-
-
1.14.13.23 3-hydroxybenzoate + NADPH + H+ + O2
-
Comamonas testosteroni 3,4-dihydroxybenzoate + NADP+ + H2O
-
?
1.14.13.23 additional information enzyme 3HB4H catalyzes an ortho-hydroxylation reaction Comamonas testosteroni ?
-
-
1.14.13.24 3-hydroxybenzoate + NADH + H+ + O2
-
Rhodococcus jostii 2,5-dihydroxybenzoate + NAD+ + H2O
-
?
1.14.13.64 4-hydroxybenzoate + NADH + 2 H+ + O2
-
Candida parapsilosis hydroquinone + NAD+ + H2O + CO2
-
?
1.14.13.64 4-hydroxybenzoate + NADH + 2 H+ + O2
-
Candida parapsilosis CBS604 hydroquinone + NAD+ + H2O + CO2
-
?
1.14.13.64 additional information 4HB1H catalyzes an oxidative decarboxylation reaction. 4HB1H is active with a wide range of 4-hydroxybenzoate (4-HB) derivatives. 4HB1H can also be induced by protocatechuate (PCA) and convert this compound directly into the central intermediate HHQ Candida parapsilosis ?
-
-
1.14.13.64 additional information 4HB1H catalyzes an oxidative decarboxylation reaction. 4HB1H is active with a wide range of 4-hydroxybenzoate (4-HB) derivatives. 4HB1H can also be induced by protocatechuate (PCA) and convert this compound directly into the central intermediate HHQ Candida parapsilosis CBS604 ?
-
-
1.14.13.64 protocatechuate + NADH + 2 H+ + O2
-
Candida parapsilosis hydroquinone + NAD+ + H2O + CO2
-
?
1.14.13.64 protocatechuate + NADH + 2 H+ + O2
-
Candida parapsilosis CBS604 hydroquinone + NAD+ + H2O + CO2
-
?
1.14.13.64 vanillate + NADH + 2 H+ + O2
-
Candida parapsilosis 3-methoxy-1,4-dihydroxybenzene + NAD+ + H2O + CO2
-
?
1.14.13.64 vanillate + NADH + 2 H+ + O2
-
Candida parapsilosis CBS604 3-methoxy-1,4-dihydroxybenzene + NAD+ + H2O + CO2
-
?

Subunits

EC Number Subunits Comment Organism
1.14.13.2 homodimer each subunit of the dimeric enzyme contains a split FAD-binding domain and a split substrate-binding domain Aspergillus niger
1.14.13.2 More structure modeling of the PhhA subunit, the FAD-binding domain, the FAD cofactor, the substrate-binding domain, the thioredoxin domain, and substrate 4-hydroxybenzoate, overview Aspergillus niger
1.14.13.64 monomer
-
Candida parapsilosis

Synonyms

EC Number Synonyms Comment Organism
1.14.13.2 4-hydroxybenzoate 3-hydroxylase
-
Aspergillus niger
1.14.13.2 An_PhhA
-
Aspergillus niger
1.14.13.2 FAD-dependent 4-hydroxybenzoate hydroxylase
-
Aspergillus niger
1.14.13.2 fungal 4-hydroxybenzoate 3-hydroxylase
-
Aspergillus niger
1.14.13.2 PHBH
-
Aspergillus niger
1.14.13.2 phhA
-
Aspergillus niger
1.14.13.23 3-hydroxybenzoate 4-hydroxylase
-
Comamonas testosteroni
1.14.13.23 3HB4H
-
Comamonas testosteroni
1.14.13.23 FAD-dependent 4-hydroxybenzoate hydroxylase
-
Comamonas testosteroni
1.14.13.23 MobA
-
Comamonas testosteroni
1.14.13.24 3-hydroxybenzoate 6-hydroxylase
-
Rhodococcus jostii
1.14.13.24 3HB6H
-
Rhodococcus jostii
1.14.13.64 4-hydroxybenzoate 1-hydroxylase (decarboxylating)
-
Candida parapsilosis
1.14.13.64 4HB1H
-
Candida parapsilosis
1.14.13.64 Cp_4HB1H
-
Candida parapsilosis
1.14.13.64 FAD-dependent 4-hydroxybenzoate hydroxylase
-
Candida parapsilosis
1.14.13.64 PHBH
-
Candida parapsilosis
1.14.13.64 V1H
-
Candida parapsilosis
1.14.13.64 vanillate 1-hydroxylase
-
Candida parapsilosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.2 25
-
assay at Aspergillus niger
1.14.13.64 25
-
-
Candida parapsilosis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.64 10
-
4-hydroxybenzoate recombinant enzyme, pH 7.6, 25°C Candida parapsilosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.2 7.6
-
assay at Aspergillus niger
1.14.13.64 7.6
-
-
Candida parapsilosis

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.2 FAD each subunit of the dimeric enzyme contains a split FAD-binding domain Aspergillus niger
1.14.13.2 NADPH
-
Aspergillus niger
1.14.13.23 FAD
-
Comamonas testosteroni
1.14.13.23 NADPH
-
Comamonas testosteroni
1.14.13.24 FAD
-
Rhodococcus jostii
1.14.13.24 NADH
-
Rhodococcus jostii
1.14.13.64 FAD addition of FAD is essential to achieve optimal turnover Candida parapsilosis
1.14.13.64 additional information 4HB1H does not contain a lipid cofactor Candida parapsilosis
1.14.13.64 NADH highly preferred cofactor Candida parapsilosis

Expression

EC Number Organism Comment Expression
1.14.13.2 Aspergillus niger the enzyme is induced by 4-hydroxybenzoate as carbon source up

General Information

EC Number General Information Comment Organism
1.14.13.2 evolution evolutionary relationship between the NAD(P)H-dependent FAD-containing 4-hydroxybenzoate hydroxylases and phylogenetic analysis of group A FPMOs, overview Aspergillus niger
1.14.13.2 evolution phylogenetic analysis shows that FAD-dependent 4-hydroxybenzoate hydroxylases reside in distinct clades of the group A flavoprotein monooxygenase (FPMO) family, indicating their separate divergence from a common ancestor. Protein homology modeling reveals that the fungal 4-hydroxybenzoate 3-hydroxylase PhhA is structurally related to phenol hydroxylase (PHHY) and 3-hydroxybenzoate 4-hydroxylase (3HB4H). 4-Hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Group A FPMOs are involved in the aerobic microbial catabolism of 4-hydroxybenzoate. Phylogenetic analysis and structure comparisons, detailed overview Aspergillus niger
1.14.13.2 metabolism enzyme PHBH is almost exclusively found in prokaryotes, where its induction, usually as a consequence of lignin degradation, results in the regioselective formation of protocatechuate, one of the central intermediates in the global carbon cycle Aspergillus niger
1.14.13.2 additional information enzyme protein homology modeling of An_PhhA using the structure file of Tc_PHHY (PDB ID 1pn0) as template, overview. Hydroxylase enzymes structure comparisons, overview Aspergillus niger
1.14.13.2 physiological function 4-hydroxybenzoate (4-HB) is a common intermediate in lignin degradation. It is one of the aromatic acids that arise from the Calpha-Cbeta cleavage of lignin components. In aerobic bacteria, 4-HB usually is converted to the ring-fission substrate 3,4-dihydroxybenzoate (protocatechuate, PCA). This reaction is catalyzed by the NAD(P)H-dependent flavoprotein monooxygenase (FPMO) 4-hydroxybenzoate 3-hydroxylase (PHBH) Aspergillus niger
1.14.13.23 evolution phylogenetic analysis of FAD-dependent 4-hydroxybenzoate hydroxylases and 3-hydroxybenzoate 4-hydroxylase, phylogenetic analysis and tree, overview. Enzyme structure analysis and comparisons (PDB ID pdb: 2dkh). Enzyme 3HB4H is missing a flexible loop, which is involved in flavin movement and closing-off the active site. In 3HB4H, the conserved tyrosine 222 makes a hydrogen bond with the hydroxyl group of the phenolic substrate, substrate binding pocket and structure-function analysis, homology modeling, overview. Residues Asp75 and Tyr271 might enhance the electron donating capacity of the hydroxyl group of 3-hydroxybenzoate (3-HB). The carboxyl group of 3-HB preferentially interacts with the side chains of His135 and Lys247. This ionic interaction is proposed to determine the orientation of bound substrate Comamonas testosteroni
1.14.13.23 additional information FAD-dependent hydroxybenzoate hydroxylase enzymes structure comparisons, overview Comamonas testosteroni
1.14.13.24 evolution evolutionary relationship between the NAD(P)H-dependent FAD-containing 4-hydroxybenzoate hydroxylases and phylogenetic analysis of group A FPMOs, overview Rhodococcus jostii
1.14.13.24 additional information 3-hydroxybenzoate 6-hydroxylase (3HB6H) structure analysis and comparison with structures of several FAD-dependent 4-hydroxybenzoate hydroxylases, homology modeling, detailed overview. FAD-dependent hydroxybenzoate hydroxylase enzymes structure comparisons Rhodococcus jostii
1.14.13.64 evolution phylogenetic analysis of FAD-dependent 4-hydroxybenzoate hydroxylases and 3-hydroxybenzoate 4-hydroxylase, phylogenetic analysis and tree, overview. Enzyme 4HB1H is only present in a small group of Saccharomycetes. Many FAD-binding proteins with unknown function in pezizomycetes and agaricomycetes have structural features in common with 4HB1H, but lack its C-terminal tail. 4HB1H belongs to a subgroup of structurally related group A FPMOs that catalyze para-hydroxylation reactions Candida parapsilosis
1.14.13.64 metabolism the enzyme is important in lignin degradation. The conversion of 4-hydroxybenzoate (4-HB) involves an ipso-attack of the electrophilic flavin C4a-hydroperoxide at the C1-atom of the activated substrate, resulting in a tetrahedral benzoquinone species and subsequent release of the carboxyl side chain. The second step of the catabolism of 4-HB in Candida parapsilosis catalyzed by the homodimeric hydroquinone hydroxylase, HQH Candida parapsilosis
1.14.13.64 additional information structure comparisons and protein homology modelling, generation of a model of Candida parapsilosis Cp_HQH and models for Cp_4HB1H, Fusarium oxysporum Fo_4HB1H-like, and Boreostereum vibrans Bv_VibMO1 using Cutaneotrichosporon cutaneum Tc_PHHY (PDB ID 1pn0), Rhodococcus jostii Rj_3HB6H (PDB ID 4bk1) and Pseudomonas putida Pp_SALH (PDB ID 5evy) as template structures, overview Candida parapsilosis
1.14.13.64 physiological function 4-hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Genome mining suggests that the 4HB1H activity is widespread in the fungal kingdom and might be responsible for the oxidative decarboxylation of vanillate, an import intermediate in lignin degradation Candida parapsilosis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.64 1000
-
4-hydroxybenzoate recombinant enzyme, pH 7.6, 25°C Candida parapsilosis