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IUBMB CommentsThe enzyme is involved in phenazine biosynthesis. The product probably spontaneously dimerises to 1,4,5a,6,9,10a-hexahydrophenazine-1,6-dicarboxylate
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(5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylic acid
(1R,6S)-6-amino-5-hydroxycyclohexa-2,4-diene-1-carboxylic acid
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(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid. The enzyme is involved in phenazine synthesis
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
charactierzation of the product by NMR spectrometry. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate45 to abstract a proton from C3 of the substrate. The proposed mechanism involves proton abstraction from C3 of trans-2,3-dihydro-3-hydroxyanthranilate by Glu45, followed by rearrangement of the double bonds and reprotonation at C1. The function of E45 as a proton shuttle is supported by results from NMR spectroscopy
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(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
PhzF is capable of producing phenazine-1-carboxylic acid, albeit slowly, from trans-2,3-dihydro-3-hydroxyanthranilic acid (i.e. (5S,6R)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxylic acid). These observations suggest that PhzF catalyzes the initial step in the conversion of trans-2,3-dihydro-3-hydroxyanthranilic acid to phenazine-1-carboxylic acid, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of trans-2,3-dihydro-3-hydroxyanthranilic acid, and that subsequent steps can occur spontaneously. A hypothetical model for how trans-2,3-dihydro-3-hydroxyanthranilic acid binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
PhzF is capable of producing phenazine-1-carboxylic acid, albeit slowly, from trans-2,3-dihydro-3-hydroxyanthranilic acid (i.e. (5S,6R)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxylic acid). These observations suggest that PhzF catalyzes the initial step in the conversion of trans-2,3-dihydro-3-hydroxyanthranilic acid to phenazine-1-carboxylic acid, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of trans-2,3-dihydro-3-hydroxyanthranilic acid, and that subsequent steps can occur spontaneously. A hypothetical model for how trans-2,3-dihydro-3-hydroxyanthranilic acid binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
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(5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylic acid
(1R,6S)-6-amino-5-hydroxycyclohexa-2,4-diene-1-carboxylic acid
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-
?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid. The enzyme is involved in phenazine synthesis
i.e. 2,3-dihydro-3-hydroxy-anthranilate
-
?
(5S,6S)-6-amino-5-hydroxycyclohexane-1,3-diene-1-carboxyate
(1R,6S)-6-amino-5-oxocyclohex-2-ene-1-carboxylate
i.e. trans-2,3-dihydro-3-hydroxyanthranilic acid
i.e. 2,3-dihydro-3-hydroxy-anthranilate
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?
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1.8 A crystal structure of PhzF, hanging drop vapor diffusion method
hanging-drop vapor-diffusion method. Crystals of unliganded PhzF belong to space group P3(2)21 with a a = b = 56 A, and c = 156 A and hold one monomer per asymmetric unit. Complexes of PhzF (with 3-hydroxyanthranilic acid and trans-2,3-dihydro-3-hydroxyanthranilic acid) crystallize in space group P2(1)2(1)2 with cell parameters of a = 93 A, b = 100 A, and c = 57 A and contain one dimer in the asymmetric unit
hanging-drop vapour-diffusion method. Crystallized from PEG 4000/ammonium sulfate/sodium citrate pH 5.6. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 56.3, c = 156.4 A. They contain one monomer in the asymmetric unit and diffract to better than 1.7 A on synchrotron beamlines. Crystals of seleno-L-methionine-labelled PhzF have been obtained
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Mavrodi, D.V.; Bleimling, N.; Thomashow, L.S.; Blankenfeldt, W.
The purification, crystallization and preliminary structural characterization of PhzF, a key enzyme in the phenazine-biosynthesis pathway from Pseudomonas fluorescens 2-79
Acta Crystallogr. Sect. D
60
184-186
2004
Pseudomonas fluorescens (Q51792), Pseudomonas fluorescens Feb 79 (Q51792)
brenda
Parsons, J.F.; Song, F.; Parsons, L.; Calabrese, K.; Eisenstein, E.; Ladner, J.E.
Structure and function of the phenazine biosynthesis protein PhzF from Pseudomonas fluorescens 2-79
Biochemistry
43
12427-12435
2004
Pseudomonas fluorescens (Q51792), Pseudomonas fluorescens Feb 79 (Q51792)
brenda
Blankenfeldt, W.; Kuzin, A.P.; Skarina, T.; Korniyenko, Y.; Tong, L.; Bayer, P.; Janning, P.; Thomashow, L.S.; Mavrodi, D.V.
Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens
Proc. Natl. Acad. Sci. USA
101
16431-16436
2004
Pseudomonas fluorescens (Q51792)
brenda
Liu, F.; Zhao, Y.; Wang, X.; Hu, H.; Peng, H.; Wang, W.; Wang, J.; Zhang, X.
Elucidation of enzymatic mechanism of phenazine biosynthetic protein PhzF using QM/MM and MD simulations
PLoS ONE
10
e0139081
2015
Pseudomonas fluorescens (Q51792)
brenda