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chorismate = 3-hydroxybenzoate + pyruvate
chorismate = 3-hydroxybenzoate + pyruvate
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chorismate = 3-hydroxybenzoate + pyruvate
a hydrolysis-elimination mechanism for CH-Hyg5 is observed. No external oxygen is incorporated into the Hyg5 products indicated an intramolecular mechanism for the Hyg5 reaction. A pericyclic mechanism, as described for chorismate-converting enzymes producing the 2- and 4-regioisomeric hydroxybenzoates, can also be ruled out due to the position of the hydroxyl group in 3-hydroxybenzoate. Arene oxide reaction mechansim analysis, overview
chorismate = 3-hydroxybenzoate + pyruvate
intramolecular mechanism catalyzed by enzyme Hyg5, overview. Reaction steps are: 1. the protonation of the methylene group of CHO, 2. nucleophilic attack of the activated 4-hydroxyl group, and 3. rearrangement of the arene oxide
chorismate = 3-hydroxybenzoate + pyruvate
the reaction of Hyg5 undergoes an arene oxide mechanism. First is, accompanied by the protonation of the methylene group, the C3-O8 bond cleavage, a concerted but asynchronous process with an energy barrier of 26.3 kcal/mol. The subsequent arene oxide formation step is quite rapid with an energy barrier of 3.7 kcal/mol. The last step is the selective ring-opening directed by C327. Residue E334 plays key roles in the formation and opening of the arene oxide intermediate
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3-hydroxybenzoate + pyruvate + H2O
chorismate
chorismate
3-hydroxybenzoate + pyruvate
chorismate
3-hydroxybenzoate + pyruvate + H2O
additional information
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3-hydroxybenzoate + pyruvate + H2O
chorismate
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r
3-hydroxybenzoate + pyruvate + H2O
chorismate
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r
chorismate
3-hydroxybenzoate + pyruvate
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chorismate
3-hydroxybenzoate + pyruvate
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chorismate
3-hydroxybenzoate + pyruvate
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chorismate
3-hydroxybenzoate + pyruvate
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ir
chorismate
3-hydroxybenzoate + pyruvate
substrate binding structure analysis
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chorismate
3-hydroxybenzoate + pyruvate
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chorismate
3-hydroxybenzoate + pyruvate
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?
chorismate
3-hydroxybenzoate + pyruvate
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chorismate
3-hydroxybenzoate + pyruvate + H2O
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-
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chorismate
3-hydroxybenzoate + pyruvate + H2O
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r
chorismate
3-hydroxybenzoate + pyruvate + H2O
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r
additional information
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no activity with 3,4-trans-dihydroxy-cyclohexa-1,5-diene-1-carboxylate and enoyl benzoate by enzyme Hyg5
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additional information
?
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no activity with 3-hydroxybenzoate and 4-hydroxybenzoate
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3-hydroxybenzoate + pyruvate + H2O
chorismate
chorismate
3-hydroxybenzoate + pyruvate
chorismate
3-hydroxybenzoate + pyruvate + H2O
additional information
?
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no activity with 3-hydroxybenzoate and 4-hydroxybenzoate
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?
3-hydroxybenzoate + pyruvate + H2O
chorismate
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r
3-hydroxybenzoate + pyruvate + H2O
chorismate
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r
chorismate
3-hydroxybenzoate + pyruvate
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?
chorismate
3-hydroxybenzoate + pyruvate
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-
?
chorismate
3-hydroxybenzoate + pyruvate
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?
chorismate
3-hydroxybenzoate + pyruvate
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-
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ir
chorismate
3-hydroxybenzoate + pyruvate
-
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?
chorismate
3-hydroxybenzoate + pyruvate
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?
chorismate
3-hydroxybenzoate + pyruvate
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-
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?
chorismate
3-hydroxybenzoate + pyruvate + H2O
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chorismate
3-hydroxybenzoate + pyruvate + H2O
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r
chorismate
3-hydroxybenzoate + pyruvate + H2O
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-
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r
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evolution
three subfamilies of chorismatases are described that convert chorismate into different (dihydro-)benzoate derivatives (CH-FkbO, CH-Hyg5, and CH-XanB2). The CH-FkbO and CH-Hyg5 subfamilies share the same protein fold, but employ fundamentally different reaction mechanisms, comparisons of the reaction mechanism of CH-FkbO and CH-Hyg5, and structure-function analysis, overview
malfunction
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a xanB2 deletion mutant exhibits a pleiotropic phenotype, including xanthomonadin deficiency, producing less exopolysaccharide, lower viability and H2O2 resistance, and lower virulence
malfunction
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a xanB2 deletion mutant exhibits a pleiotropic phenotype, including xanthomonadin deficiency, producing less exopolysaccharide, lower viability and H2O2 resistance, and lower virulence
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metabolism
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the enzyme is a key metabolic enzyme linking xanthomonadin, coenzyme Q, and exopolysaccharide biosynthesis
metabolism
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the enzyme is a key metabolic enzyme linking xanthomonadin, coenzyme Q, and exopolysaccharide biosynthesis
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additional information
analysis of catalytic mechanisms of chorismatases EC 3.3.2.13 and EC 4.1.3.45 by molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations of the Michaelis complexes of two wild-type models (FkbO and Hyg5) and four mutants models with chorismate as substrate, comparison of the catalytic mechanisms between FkbO and Hyg5, overview. The A/G residue group (A244FkbO/G240Hyg5) causes changes in the binding states of the substrate and the orientation of the catalytic glutamate, but only these changes affect the product selectivity in chorismatases limitedly. The distal V/Q residue group, which determines the internal water self-regulating ability at the active site, has significant impact on the selectivity of the catalytic mechanisms. The V/Q residue group is suggested to be an important factor to control the catalytic activities in chorismatases
additional information
involvement of residue C327 in directing the selectivity of product formation is underlined by the crystal structure of the mutant Hyg5 C327S with the product 3-hydroxybenzoate
additional information
the structure of Streptomyces hygroscopicus chorismatase CH-Hyg5 is very similar to that of Streptomyces hygroscopicus chorismatase CH-Fkbo, all amino acid residues in the active site are the same except residues G240Hyg5 (A244Fkb8) and C327Hyg5 (A331Fkb8) are the same. The optimized active site structure of Hyg5 complex is superpositioned with that of Fkbo, overview. Two nonconserved active site residues are responsible for the different reaction mechanism of CH-Fkbo and CH-Hyg5. In CH-Hyg5, the lack of methyl in G240Hyg5 leads to the different conformation of the side chain of E334 (or E338) in Hyg5 and Fkbo. This small change eventually decreases the ESP charge of C3 atom of the substrate, which facilitates the cleavage of C3-O8 bond in Hyg5. Furthermore, C327Hyg5 is involved in the selective product formation in Hyg5 enzyme
additional information
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the structure of Streptomyces hygroscopicus chorismatase CH-Hyg5 is very similar to that of Streptomyces hygroscopicus chorismatase CH-Fkbo, all amino acid residues in the active site are the same except residues G240Hyg5 (A244Fkb8) and C327Hyg5 (A331Fkb8) are the same. The optimized active site structure of Hyg5 complex is superpositioned with that of Fkbo, overview. Two nonconserved active site residues are responsible for the different reaction mechanism of CH-Fkbo and CH-Hyg5. In CH-Hyg5, the lack of methyl in G240Hyg5 leads to the different conformation of the side chain of E334 (or E338) in Hyg5 and Fkbo. This small change eventually decreases the ESP charge of C3 atom of the substrate, which facilitates the cleavage of C3-O8 bond in Hyg5. Furthermore, C327Hyg5 is involved in the selective product formation in Hyg5 enzyme
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C327A/G240A
the Hyg5 double mutant shows the same behavior as the single mutants, but along with a more dramatic loss of activity
Q201V
site-directed mutagenesis
C327A
the mutation leads to decreased activity (6%) compared to the wild type enzyme and results in a complete loss of product selectivity
C327A
the Hyg5 mutant shows 6% decreased activity compared to the wild-type, and results in a complete loss of product selectivity. The FkbO product (4R,5R)-4,5-dihydroxycyclohexa-1(6),2-diene-1-carboxylate as well as the XanB2 product 4-hydroxybenzoate are produced
G240A
site-directed mutagenesis
G240A
the mutation leads to decreased activity (55%) compared to the wild type enzyme and results in a complete loss of product selectivity
G240A
the Hyg5 mutant shows 55% decreased activity compared to the wild-type, and results in a complete loss of product selectivity. The FkbO product (4R,5R)-4,5-dihydroxycyclohexa-1(6),2-diene-1-carboxylate as well as the XanB2 product 4-hydroxybenzoate are produced
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Jiang, Y.; Wang, H.; Lu, C.; Ding, Y.; Li, Y.; Shen, Y.
Identification and characterization of the cuevaene A biosynthetic gene cluster in Streptomyces sp. LZ35
ChemBioChem
14
1468-1475
2013
Streptomyces sp., Streptomyces sp. LZ35
brenda
Zhou, L.; Wang, J.Y.; Wu, J.; Wang, J.; Poplawsky, A.; Lin, S.; Zhu, B.; Chang, C.; Zhou, T.; Zhang, L.H.; He, Y.W.
The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways
Mol. Microbiol.
87
80-93
2013
Xanthomonas campestris pv. campestris
brenda
Zhou, L.; Huang, T.W.; Wang, J.Y.; Sun, S.; Chen, G.; Poplawsky, A.; He, Y.W.
The rice bacterial pathogen Xanthomonas oryzae pv. oryzae produces 3-hydroxybenzoic acid and 4-hydroxybenzoic acid via XanB2 for use in xanthomonadin, ubiquinone, and exopolysaccharide biosynthesis
Mol. Plant Microbe Interact.
26
1239-1248
2013
Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzae PXO99A
brenda
Andexer, J.N.; Kendrew, S.G.; Nur-e-Alam, M.; Lazos, O.; Foster, T.A.; Zimmermann, A.S.; Warneck, T.D.; Suthar, D.; Coates, N.J.; Koehn, F.E.; Skotnicki, J.S.; Carter, G.T.; Gregory, M.A.; Martin, C.J.; Moss, S.J.; Leadlay, P.F.; Wilkinson, B.
Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate
Proc. Natl. Acad. Sci. USA
108
4776-4781
2011
Streptomyces hygroscopicus
brenda
Hubrich, F.; Juneja, P.; Mueller, M.; Diederichs, K.; Welte, W.; Andexer, J.N.
Chorismatase mechanisms reveal fundamentally different types of reaction in a single conserved protein fold
J. Am. Chem. Soc.
137
11032-11037
2015
Streptomyces hygroscopicus (O30478)
brenda
Schoener, T.A.; Fuchs, S.W.; Reinhold-Hurek, B.; Bode, H.B.
Identification and biosynthesis of a novel xanthomonadin-dialkylresorcinol-hybrid from Azoarcus sp. BH72
PLoS ONE
9
e90922
2014
Azoarcus olearius
brenda
Zhang, Y.; Zhang, H.; Zheng, Q.
How chorismatases regulate distinct reaction channels in a single conserved active pocket mechanistic analysis with QM/MM (ONIOM) investigations
Chemistry
25
1326-1336
2019
Streptomyces hygroscopicus (O30478)
brenda
Dong, L.; Liu, Y.
Comparative studies of the catalytic mechanisms of two chorismatases CH-fkbo and CH-Hyg5
Proteins
85
1146-1158
2017
Streptomyces hygroscopicus (O30478), Streptomyces hygroscopicus
brenda
4.1.3.45 3-hydroxybenzoate synthase
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