1.14.20.15: L-threonyl-[L-threonyl-carrier protein] 4-chlorinase
This is an abbreviated version!
For detailed information about L-threonyl-[L-threonyl-carrier protein] 4-chlorinase, go to the full flat file.
Word Map on EC 1.14.20.15
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1.14.20.15
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non-heme
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ferryl
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rebound
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halide
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alphakg-dependent
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unactivated
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chemoselectivity
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alphakg
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ironii
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high-spin
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alpha-ketoglutarate-dependent
- 1.14.20.15
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non-heme
-
ferryl
-
rebound
- halide
-
alphakg-dependent
-
unactivated
-
chemoselectivity
-
alphakg
-
ironii
-
high-spin
-
alpha-ketoglutarate-dependent
Reaction
Synonyms
aliphatic halogenase, SyrB2, syringomycin biosynthesis enzyme 2, Thr3
ECTree
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Substrates Products
Substrates Products on EC 1.14.20.15 - L-threonyl-[L-threonyl-carrier protein] 4-chlorinase
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REACTION DIAGRAM
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + 2 Cl-
4,4-dichloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Br-
4-bromo-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
4,4-dichloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + 2 Cl-
4,4-dichloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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?
4-bromo-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Br-
4-bromo-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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?
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the enzyme participates in syringomycin E biosynthesis
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
substrate positioning controls the partition between halogenation and hydroxylation
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the enzyme can not chlorinate free L-Thr or the small molecule surrogate for L-Thr-S-SyrB1, L-Thr-S-N-acetylcysteamine
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the substrate consists of L-Thr tethered via thioester linkage to a covalently bound phosphopantetheine cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free L-Thr or its analogues, but SyrB1 charged either by L-Thr or by any of several non-native amino acids does trigger the reaction by as much as 8000fold (for L-Thr-S-SyrB1). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-20fold when the native L-Thr is replaced by another amino acid and by about 40fold when SyrB1 is replaced by a heterologous carrier protein, CytC2. The SyrB2 chloroferryl state exhibits unprecedented stability (t1/2 = 30-110 min at 0°C), can be trapped in high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization
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L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the enzyme participates in syringomycin E biosynthesis
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-
?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the substrate consists of L-Thr tethered via thioester linkage to a covalently bound phosphopantetheine cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free L-Thr or its analogues, but SyrB1 charged either by L-Thr or by any of several non-native amino acids does trigger the reaction by as much as 8000fold (for L-Thr-S-SyrB1). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-20fold when the native L-Thr is replaced by another amino acid and by about 40fold when SyrB1 is replaced by a heterologous carrier protein, CytC2. The SyrB2 chloroferryl state exhibits unprecedented stability (t1/2 = 30-110 min at 0°C), can be trapped in high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
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?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the enzyme participates in syringomycin E biosynthesis
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-
?
L-threonyl-[L-threonyl-carrier protein SyrB1] + 2-oxoglutarate + O2 + Cl-
4-chloro-L-threonyl-[L-threonyl-carrier protein SyrB1] + succinate + CO2 + H2O
the enzyme can not chlorinate free L-Thr or the small molecule surrogate for L-Thr-S-SyrB1, L-Thr-S-N-acetylcysteamine
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the enzyme catalyzes chlorination of the C4 position of L-threonine appended via a thioester linkage to the phosphopantetheine arm of the companion aminoacyl carrier protein, SyrB1. The native substrate of SyrB2, Thr, is almost exclusively chlorinated, although non-native substrates undergo hydroxylation as well as chlorination. SyrB2 represents an intriguing case in which two different reaction outcomes catalyzed by this enzyme family (hydroxylation and halogenation) are observed
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additional information
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non-native substrates undergo hydroxylation as well as chlorination. The cis-chloroferryl complex in enzyme SyrB2 reacts more rapidly with SyrB1 presenting L-aminobutyric acid or L-norvaline than with L-threonine. Selectivity for chlorination is also strongly modulated: L-threonine is almost exclusively chlorinated, L-aminobutyric acid is chlorinated and hydroxylated at C4 to similar extents, and L-norvaline is predominately hydroxylated at the C5 position. The differential reactivity observed for the different substrates might arise primarily from substrate-protein interactions that impact the partition between the axial and equatorial coordination isomers of the ferryl complex rather than from substrate positioning per se
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additional information
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the enzyme catalyzes chlorination of the C4 position of L-threonine appended via a thioester linkage to the phosphopantetheine arm of the companion aminoacyl carrier protein, SyrB1. The native substrate of SyrB2, Thr, is almost exclusively chlorinated, although non-native substrates undergo hydroxylation as well as chlorination. SyrB2 represents an intriguing case in which two different reaction outcomes catalyzed by this enzyme family (hydroxylation and halogenation) are observed
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additional information
?
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non-native substrates undergo hydroxylation as well as chlorination. The cis-chloroferryl complex in enzyme SyrB2 reacts more rapidly with SyrB1 presenting L-aminobutyric acid or L-norvaline than with L-threonine. Selectivity for chlorination is also strongly modulated: L-threonine is almost exclusively chlorinated, L-aminobutyric acid is chlorinated and hydroxylated at C4 to similar extents, and L-norvaline is predominately hydroxylated at the C5 position. The differential reactivity observed for the different substrates might arise primarily from substrate-protein interactions that impact the partition between the axial and equatorial coordination isomers of the ferryl complex rather than from substrate positioning per se
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additional information
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computational study of reaction at a model complex of the SyrB2 enzyme active site. The first step, alpha-ketoglutarate decarboxylation, is barrierless and exothermic, while the subsequent hydrogen abstraction step has an energetic barrier consistent with that accessible under biological conditions. The hydrogen abstraction and radical chlorination steps are strongly coupled: the barrier for the hydrogen abstraction step is reduced when carried out concomitantly with the exothermic chlorination step
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additional information
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mechanism of the chlorination reaction of SyrB2 is studied with computational methods. The structure of the SyrB2-substrate complex is modeled with the use of molecular docking procedures. DFT calculations performed with a model involving all first-shell and several second-shell ligands of iron provide energy profiles, which suggest that the two forms of the oxoferryl species can both participate in the reaction. Relative energies of transition states for C-H bond cleavage by these two reactive oxoferryl species dictate the product specificity. The identity of the two oxoferryl species observed in the experimental works is proposed and confirmed by theoretical calculations of their Mössbauer isomer shifts and quadrupole splittings. CASPT2 energy calculations for the oxoferryl species in the quintet, triplet, and septet spin states, together with the DFT results for the reaction pathway, suggest that once the Fe(IV)-O species is formed, the reaction proceeds exclusively on the quintet potential energy surface
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additional information
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the ability of an Fe(IV)-O intermediate in SyrB2 to perform chlorination vs. hydroxylation is computationally evaluated for different substrates. Differential contribution of the two frontier molecular orbitals to chlorination vs. hydroxylation selectivity in SyrB2 is related to a reaction mechanism that involves two asynchronous transfers: electron transfer from the substrate radical to the iron center followed by late ligand (Cl- or OH-) transfer to the substrate
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additional information
?
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computational study of reaction at a model complex of the SyrB2 enzyme active site. The first step, alpha-ketoglutarate decarboxylation, is barrierless and exothermic, while the subsequent hydrogen abstraction step has an energetic barrier consistent with that accessible under biological conditions. The hydrogen abstraction and radical chlorination steps are strongly coupled: the barrier for the hydrogen abstraction step is reduced when carried out concomitantly with the exothermic chlorination step
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?
additional information
?
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mechanism of the chlorination reaction of SyrB2 is studied with computational methods. The structure of the SyrB2-substrate complex is modeled with the use of molecular docking procedures. DFT calculations performed with a model involving all first-shell and several second-shell ligands of iron provide energy profiles, which suggest that the two forms of the oxoferryl species can both participate in the reaction. Relative energies of transition states for C-H bond cleavage by these two reactive oxoferryl species dictate the product specificity. The identity of the two oxoferryl species observed in the experimental works is proposed and confirmed by theoretical calculations of their Mössbauer isomer shifts and quadrupole splittings. CASPT2 energy calculations for the oxoferryl species in the quintet, triplet, and septet spin states, together with the DFT results for the reaction pathway, suggest that once the Fe(IV)-O species is formed, the reaction proceeds exclusively on the quintet potential energy surface
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?
additional information
?
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the ability of an Fe(IV)-O intermediate in SyrB2 to perform chlorination vs. hydroxylation is computationally evaluated for different substrates. Differential contribution of the two frontier molecular orbitals to chlorination vs. hydroxylation selectivity in SyrB2 is related to a reaction mechanism that involves two asynchronous transfers: electron transfer from the substrate radical to the iron center followed by late ligand (Cl- or OH-) transfer to the substrate
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?