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tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
reaction mechanism
-
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
FADH2 and O2 form an 4alpha-FAD-OOH intermediate that decays to 4alpha-FAD-OH. Tryptophan likely does not react directly with any flavin intermediate. Substrate chlorination occurs after completion of the flavin redox reactions
-
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. the resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution
-
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
monooxygenase-like mechanism proposed. First, FADH2 is produced by a flavin reductase using NADH. FADH2 binds to flavin-free enzyme and reacts to enzyme-bound 4alpha-flavin hydroperoxide which attacks L-tryptophan. Activated tryptophan is attacked by chloride as a nucleophile, the resulting halohydrin is dehydrated to the end product 7-chlorotryptophan
-
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
proposed mechanism: after formation of a FAD-OOH intermediate, the reactive chlorine is generated as a FAD-O-Cl intermediate. Chlorination of L-tryptophan proceeds by attack of the aromatic pi electrons on the intermediate in a two-electron mechanism and abstraction of a proton generates the final product
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
reaction mechanism, combined quantum mechanical/molecular mechanical (QM/MM) calculations of potential energy and free energy surfaces, using the structure, PDB ID 2AR8, and employing density functional theory. First reaction step is the formation of an arenium ion (Wheland intermediate), the second reaction step is the deprotonation of the Wheland intermediate by residue E346 oxygen, at the final stage of the mechanism residues K79 and E346 are regenerated
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
proposed mechanism: after formation of a FAD-OOH intermediate, the reactive chlorine is generated as a FAD-O-Cl intermediate. Chlorination of L-tryptophan proceeds by attack of the aromatic pi electrons on the intermediate in a two-electron mechanism and abstraction of a proton generates the final product
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-
tryptophan + FADH2 + chloride + O2 + H+ = 7-chloro-L-tryptophan + FAD + 2 H2O
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5-amino-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-amino-L-tryptophan + FAD + 2 H2O
-
-
-
?
5-bromo-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-bromo-L-tryptophan + FAD + 2 H2O
-
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-
?
5-fluoro-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-fluoro-L-tryptophan + FAD + 2 H2O
-
-
-
?
5-hydroxy-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-hydroxy-L-tryptophan + FAD + 2 H2O
5-methyl-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-methyl-L-tryptophan + FAD + 2 H2O
-
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-
?
6-chloro-L-tryptophan + FADH2 + chloride + O2 + H+
6,7-dichloro-L-tryptophan + FAD + 2 H2O
D-tryptophan + FADH2 + bromide + O2 + H+
7-bromo-D-tryptophan + FAD + 2 H2O
regioselective halogenation, recombinant enzyme
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-
?
L-tryptophan + FADH2 + Br- + O2 + H+
7-bromo-L-tryptophan + FAD + H2O
L-tryptophan + FADH2 + bromide + O2 + H+
7-bromo-L-tryptophan + FAD + 2 H2O
best substrates, regioselective halogenation, recombinant enzyme
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-
?
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
L-tryptophan + FADH2 + Cl- + O2
7-chloro-L-tryptophan + FAD + H2O
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
tryptamine + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptamine + FAD + H2O
-
-
-
?
tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
tryptophan + FADH2 + O2 + Cl- + H+
7-chlorotryptophan + FAD + H2O
-
initial step of rebeccamycin biosynthesis
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-
?
additional information
?
-
5-hydroxy-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-hydroxy-L-tryptophan + FAD + 2 H2O
-
-
-
?
5-hydroxy-L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-5-hydroxy-L-tryptophan + FAD + 2 H2O
regioselective halogenation, recombinant enzyme
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-
?
6-chloro-L-tryptophan + FADH2 + chloride + O2 + H+
6,7-dichloro-L-tryptophan + FAD + 2 H2O
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-
-
?
6-chloro-L-tryptophan + FADH2 + chloride + O2 + H+
6,7-dichloro-L-tryptophan + FAD + 2 H2O
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-
?
L-tryptophan + FADH2 + Br- + O2 + H+
7-bromo-L-tryptophan + FAD + H2O
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-
?
L-tryptophan + FADH2 + Br- + O2 + H+
7-bromo-L-tryptophan + FAD + H2O
-
identity of product is confirmed by ESI-MS and 1H-NMR. Reaction is selective for the 7-position. Brominating activity is about 75% of chlorinating activity
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?
L-tryptophan + FADH2 + Br- + O2 + H+
7-bromo-L-tryptophan + FAD + H2O
-
identity of product is confirmed by ESI-MS and 1H-NMR. Reaction is selective for the 7-position. Brominating activity is about 75% of chlorinating activity
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?
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
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-
?
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2
7-chloro-L-tryptophan + FAD + H2O
-
formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin
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?
L-tryptophan + FADH2 + Cl- + O2
7-chloro-L-tryptophan + FAD + H2O
-
reaction of FADH2, Cl-, and O2 in the active site, involving active site Lys79, generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction, formation of a long-living chlorinating intermediate, which remains on the enzyme after removal of FAD and transfers chlorine to tryptophan with kinetically competent rates, substrate binding structure, overview
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?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
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-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
identity of product is confirmed by ESI-MS and 1H-NMR
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
identity of product is confirmed by ESI-MS and 1H-NMR
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
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?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
regioselective reaction
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?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
the enzyme is involved in biosynthesis of pyrrolnitrin
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-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
the enzyme is involved in biosynthesis of pyrrolnitrin
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
rebeccamycin biosynthesis
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
chlorination of its natural substrate tryptophan exclusively at the 7-position, a position at which direct chemical chlorination is not possible
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-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
rebeccamycin biosynthesis
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
chlorination of its natural substrate tryptophan exclusively at the 7-position, a position at which direct chemical chlorination is not possible
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?
additional information
?
-
KtzQ shows no activity for chlorination of piperazic acid or gamma,delta-dehydropiperazic acid but is active when incubated with L-Trp. Substrate specificity of KtzQ in a coupled assay system with flavin reductase KtzS, overview. Product analysis with NMR spectroscopy and ESI-mass spectrometry. KtzQ is a regiospecific tryptophan-7-halogenase
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?
additional information
?
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first step in biosynthesis of rebeccamycin
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?
additional information
?
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O2 and presence of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase, are required for reaction. 54% of available L-tryptophan are converted within 5 min when the reaction is prepapred anaerobically and O2 is introduced slowly. Reaction rate drops by 90% in air-saturated conditions. No substrate: fluoride, iodide
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?
additional information
?
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a combination of tryptophan synthase from Salmonella enterica and tryptophan-7-halogenase RebH from Lechevalieria aerocolonigenes for the regioselective halogenation of substituted tryptophan derivatives. An array of C5- and C6-substituted tryptophan derivatives is synthesized and 7-halogenated by RebH. Halogenation products of tryptophan derivatives by RebH, overview. Halogenation occurs in many cases preferably in the electronically unfavored C7 position, even in presence of deactivating ortho/para-directing groups like 5-fluoro
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?
additional information
?
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O2 and presence of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase, are required for reaction. 54% of available L-tryptophan are converted within 5 min when the reaction is prepapred anaerobically and O2 is introduced slowly. Reaction rate drops by 90% in air-saturated conditions. No substrate: fluoride, iodide
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?
additional information
?
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first step in pyrrolnitrin biosynthetic pathway
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?
additional information
?
-
Trp 7-hal can accept a number of different tryptophan, indole, and phenylpyrrole derivatives (e.g. 3-(2'-aminophenyl)pyrrole or monodechloroaminopyrrolnitrin), but only tryptophan is regioselectively chlorinated in the 7-position of the indole ring. With all the other compounds, the reaction proceeds with a relaxed regioselectivity
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?
additional information
?
-
Trp 7-hal can accept a number of different tryptophan, indole, and phenylpyrrole derivatives (e.g. 3-(2'-aminophenyl)pyrrole or monodechloroaminopyrrolnitrin), but only tryptophan is regioselectively chlorinated in the 7-position of the indole ring. With all the other compounds, the reaction proceeds with a relaxed regioselectivity
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?
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6-chloro-L-tryptophan + FADH2 + chloride + O2 + H+
6,7-dichloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
L-tryptophan + FADH2 + Cl- + O2
7-chloro-L-tryptophan + FAD + H2O
-
formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
tryptophan + FADH2 + O2 + Cl- + H+
7-chlorotryptophan + FAD + H2O
-
initial step of rebeccamycin biosynthesis
-
-
?
additional information
?
-
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + chloride + O2 + H+
7-chloro-L-tryptophan + FAD + 2 H2O
-
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
-
the enzyme is involved in biosynthesis of pyrrolnitrin
-
-
?
L-tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-L-tryptophan + FAD + H2O
the enzyme is involved in biosynthesis of pyrrolnitrin
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
rebeccamycin biosynthesis
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
chlorination of its natural substrate tryptophan exclusively at the 7-position, a position at which direct chemical chlorination is not possible
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
rebeccamycin biosynthesis
-
-
?
tryptophan + FADH2 + Cl- + O2 + H+
7-chloro-tryptophan + FAD + H2O
chlorination of its natural substrate tryptophan exclusively at the 7-position, a position at which direct chemical chlorination is not possible
-
-
?
additional information
?
-
-
first step in biosynthesis of rebeccamycin
-
-
?
additional information
?
-
-
first step in pyrrolnitrin biosynthetic pathway
-
-
?
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Dong, C.; Kotzsch, A.; Dorward, M.; van Pee, K.H.; Naismith, J.H.
Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens
Acta Crystallogr. Sect. D
60
1438-1440
2004
Pseudomonas fluorescens
brenda
Unversucht, S.; Hollmann, F.; Schmid, A.; van Pee, K.
FADH2-dependence of tryptophan 7-halogenase
Adv. Synth. Catal.
347
1163-1167
2005
Pseudomonas fluorescens
-
brenda
Yeh, E.; Cole, L.J.; Barr, E.W.; Bollinger, J.M.; Ballou, D.P.; Walsh, C.T.
Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH
Biochemistry
45
7904-7912
2006
Lentzea aerocolonigenes
brenda
Yeh, E.; Garneau, S.; Walsh, C.T.
Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis
Proc. Natl. Acad. Sci. USA
102
3960-3965
2005
Lentzea aerocolonigenes (Q8KHZ8), Lentzea aerocolonigenes 39243 (Q8KHZ8)
brenda
Dong, C.; Flecks, S.; Unversucht, S.; Haupt, C.; van Pee, K.H.; Naismith, J.H.
Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination
Science
309
2216-2219
2005
Pseudomonas fluorescens
brenda
Yeh, E.; Blasiak, L.C.; Koglin, A.; Drennan, C.L.; Walsh, C.T.
Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases
Biochemistry
46
1284-1292
2007
Lentzea aerocolonigenes
brenda
Hlzer, M.; Burd, W.; Reiig, H.U.; van pee, K.-H.
Substrate specificity and regioselectivity of tryptophan 7-halogenase from Pseudomonas fluorescens BL915
Adv. Synth. Catal.
343
591-595
2001
Pseudomonas fluorescens (Q9RPG3), Pseudomonas fluorescens BL915 (Q9RPG3)
-
brenda
Keller, S.; Wage, T.; Hohaus, K.; Hlzer, M.; Eichhorn, E.; van Pee, K.H.
Purification and Partial Characterization of Tryptophan 7-Halogenase (PrnA) from Pseudomonas fluorescens
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39
2300-2302
2000
Pseudomonas fluorescens
brenda
Hammer, P.E.; Hill, D.S.; Lam, S.T.; van Pee, K.H.; Ligon, J.M.
Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin
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63
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1997
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brenda
van Pee, K.H.; Patallo, E.P.
Flavin-dependent halogenases involved in secondary metabolism in bacteria
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70
631-641
2006
Lentzea aerocolonigenes (Q8KHZ8), Pseudomonas fluorescens (Q4KAM7)
brenda
Kirner, S.; Hammer, P.E.; Hill, D.S.; Altmann, A.; Fischer, I.; Weislo, L.J.; Lanahan, M.; van Pee, K.H.; Ligon, J.M.
Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens
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180
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1998
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The structure of flavin-dependent tryptophan 7-halogenase RebH
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70
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Bitto, E.; Huang, Y.; Bingman, C.A.; Singh, S.; Thorson, J.S.; Phillips, G.N.
The structure of flavin-dependent tryptophan 7-halogenase RebH
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70
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2008
Lentzea aerocolonigenes (Q8KHZ8)
brenda
Lang, A.; Polnick, S.; Nicke, T.; William, P.; Patallo, E.; Naismith, J.; Van Pee, K.
Changing the regioselectivity of the tryptophan 7-halogenase PrnA by site-directed mutagenesis
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50
2951-2953
2011
Pseudomonas fluorescens
brenda
Glenn, W.S.; Nims, E.; OConnor, S.E.
Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor
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133
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Frese, M.; Sewald, N.
Enzymatic halogenation of tryptophan on a gram scale
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brenda
Fraebel, S.; Krischke, M.; Staniek, A.; Warzecha, H.
Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes
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11
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Frese, M.; Guzowska, P.; Voss, H.; Sewald, N.
Regioselective enzymatic halogenation of substituted tryptophan derivatives using the FAD-dependent halogenase RebH
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6
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Lentzea aerocolonigenes (Q8KHZ8)
-
brenda
Heemstra, J.R.Jr.; Walsh, C.T.
Tandem action of the O2- and FADH2-dependent halogenases KtzQ and KtzR produce 6,7-dichlorotryptophan for Kutzneride assembly
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130
14024-14025
2008
Kutzneria sp. 744 (A8CF75)
brenda
Karabencheva-Christova, T.G.; Torras, J.; Mulholland, A.J.; Lodola, A.; Christov, C.Z.
Mechanistic insights into the reaction of chlorination of tryptophan catalyzed by tryptophan 7-halogenase
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7
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