Limits...
Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

Wong C, Fujimori DG, Walsh CT, Drennan CL - J. Am. Chem. Soc. (2009)

Bottom Line: To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution.These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron.Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.

ABSTRACT
CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

Show MeSH
Overall structure of CytC3 dimer. (a) Crystallographic dimer is colored by molecule. The iron ligands: two protein residues (His118 and His240) and αKG are shown in stick representation, and waters in the active site are shown in a spherical representation. The ends of the disordered region are indicated as “missing loop”, which consists of residues 178−219. (b) Structural alignment of SyrB2 monomer (pink) and CytC3 dimer (blue). (c) Surface representation of CytC3 dimer showing access to active site on one face of the crystallographic dimer. Active site waters are colored in red; each monomer of CytC3 is colored yellow and blue. The ends of the disordered region are colored in green.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2663892&req=5

fig2: Overall structure of CytC3 dimer. (a) Crystallographic dimer is colored by molecule. The iron ligands: two protein residues (His118 and His240) and αKG are shown in stick representation, and waters in the active site are shown in a spherical representation. The ends of the disordered region are indicated as “missing loop”, which consists of residues 178−219. (b) Structural alignment of SyrB2 monomer (pink) and CytC3 dimer (blue). (c) Surface representation of CytC3 dimer showing access to active site on one face of the crystallographic dimer. Active site waters are colored in red; each monomer of CytC3 is colored yellow and blue. The ends of the disordered region are colored in green.

Mentions: The structure of CytC3 was solved by molecular replacement using PHASER(20) with the apo CytC3 data set to 3 Å resolution. The structure of SyrB2 (2FCU), with 58% identity and 71% homology to CytC3, without any ligands or waters was used as a search model.(8) The best rotational and translational solution has a correlation coefficient of 16 with two CytC3 molecules per asymmetric unit. The resulting electron density map was solvent flattened in SHARP.(21) Side chains were added to the model at 3 Å resolution, followed by iterative rounds of model building and refinement using XtalView and CNS, respectively.22,23 Phases were extended to 2.2 Å resolution, waters were added to the model after few rounds of refinement, and further model building and refinements were done in COOT(24) and CNS, respectively. No noncrystallographic symmetry (NCS) or sigma cutoff was used during the refinement. The final apo structure was refined to 2.2 Å resolution with Rcryst = 21.8% and Rfree = 26.0%. Since the iron CytC3 crystals were isomorphous with the apo CytC3 crystals, a rigid body refinement followed by simulated annealing refinement was used. The iron structure was refined using a patch that restrains the bond lengths and angles of the ligands to the metal using higher weights for protein ligands (500 for both bond length and angle weights) and αKG (500 bond length weight and 100 angle weight), and lower weights for the water molecules (50 bond length weight and no angle weight). The final iron structure was refined to 2.2 Å resolution with Rcryst = 23.8% and Rfree = 27.8% with the same reflection test set as for the apo structure. Composite omit maps calculated using CNS simulated annealing were used to validate the models, and an anomalous difference map was used to verify the positions of the irons. For both the apo and iron structures, residues 8−317 were observed out of a total of 319 residues. There is one major chain break in both structures (residues 178−220 in molecule A, and 179−216 in molecule B), and a minor chain break only in molecule A of the apo structure (residues 170−172). Figures 2, 4−6 were generated in pymol. Figure 3 was generated in ESPript server v.2.1 (Laboratorie des Interactions Plantes-Microorganismes, France).


Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

Wong C, Fujimori DG, Walsh CT, Drennan CL - J. Am. Chem. Soc. (2009)

Overall structure of CytC3 dimer. (a) Crystallographic dimer is colored by molecule. The iron ligands: two protein residues (His118 and His240) and αKG are shown in stick representation, and waters in the active site are shown in a spherical representation. The ends of the disordered region are indicated as “missing loop”, which consists of residues 178−219. (b) Structural alignment of SyrB2 monomer (pink) and CytC3 dimer (blue). (c) Surface representation of CytC3 dimer showing access to active site on one face of the crystallographic dimer. Active site waters are colored in red; each monomer of CytC3 is colored yellow and blue. The ends of the disordered region are colored in green.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2663892&req=5

fig2: Overall structure of CytC3 dimer. (a) Crystallographic dimer is colored by molecule. The iron ligands: two protein residues (His118 and His240) and αKG are shown in stick representation, and waters in the active site are shown in a spherical representation. The ends of the disordered region are indicated as “missing loop”, which consists of residues 178−219. (b) Structural alignment of SyrB2 monomer (pink) and CytC3 dimer (blue). (c) Surface representation of CytC3 dimer showing access to active site on one face of the crystallographic dimer. Active site waters are colored in red; each monomer of CytC3 is colored yellow and blue. The ends of the disordered region are colored in green.
Mentions: The structure of CytC3 was solved by molecular replacement using PHASER(20) with the apo CytC3 data set to 3 Å resolution. The structure of SyrB2 (2FCU), with 58% identity and 71% homology to CytC3, without any ligands or waters was used as a search model.(8) The best rotational and translational solution has a correlation coefficient of 16 with two CytC3 molecules per asymmetric unit. The resulting electron density map was solvent flattened in SHARP.(21) Side chains were added to the model at 3 Å resolution, followed by iterative rounds of model building and refinement using XtalView and CNS, respectively.22,23 Phases were extended to 2.2 Å resolution, waters were added to the model after few rounds of refinement, and further model building and refinements were done in COOT(24) and CNS, respectively. No noncrystallographic symmetry (NCS) or sigma cutoff was used during the refinement. The final apo structure was refined to 2.2 Å resolution with Rcryst = 21.8% and Rfree = 26.0%. Since the iron CytC3 crystals were isomorphous with the apo CytC3 crystals, a rigid body refinement followed by simulated annealing refinement was used. The iron structure was refined using a patch that restrains the bond lengths and angles of the ligands to the metal using higher weights for protein ligands (500 for both bond length and angle weights) and αKG (500 bond length weight and 100 angle weight), and lower weights for the water molecules (50 bond length weight and no angle weight). The final iron structure was refined to 2.2 Å resolution with Rcryst = 23.8% and Rfree = 27.8% with the same reflection test set as for the apo structure. Composite omit maps calculated using CNS simulated annealing were used to validate the models, and an anomalous difference map was used to verify the positions of the irons. For both the apo and iron structures, residues 8−317 were observed out of a total of 319 residues. There is one major chain break in both structures (residues 178−220 in molecule A, and 179−216 in molecule B), and a minor chain break only in molecule A of the apo structure (residues 170−172). Figures 2, 4−6 were generated in pymol. Figure 3 was generated in ESPript server v.2.1 (Laboratorie des Interactions Plantes-Microorganismes, France).

Bottom Line: To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution.These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron.Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, Massachusetts 02139, USA.

ABSTRACT
CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

Show MeSH