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Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli.

McGregor WC, Gillner DM, Swierczek SI, Liu D, Holz RC - Springerplus (2013)

Bottom Line: The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble.Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE.Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM.

View Article: PubMed Central - PubMed

Affiliation: The Department of Applied Sciences and Mathematics, College of Technology and Innovation, Arizona State University, Mesa, AZ 85212 USA.

ABSTRACT
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

No MeSH data available.


Related in: MedlinePlus

Homology model of the [ZnZn(ArgE)] fromE. colibased on the X-ray structure of DapE. A) Overlay of the ArgE homology model (Green) and the X-ray crystal structure of DapE (Magenta). The two Zn(II) ions and the bridging water from ArgE are shown as spheres. B) Conserved active site residues for ArgE (Green) and DapE (Magenta). Nitrogen atoms are in blue, oxygen atoms are in red. The residues are labeled with single-letter amino acid codes with the labels for residues from DapE in parenthesis.
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Fig3: Homology model of the [ZnZn(ArgE)] fromE. colibased on the X-ray structure of DapE. A) Overlay of the ArgE homology model (Green) and the X-ray crystal structure of DapE (Magenta). The two Zn(II) ions and the bridging water from ArgE are shown as spheres. B) Conserved active site residues for ArgE (Green) and DapE (Magenta). Nitrogen atoms are in blue, oxygen atoms are in red. The residues are labeled with single-letter amino acid codes with the labels for residues from DapE in parenthesis.

Mentions: A three-dimensional homological structure of the ArgE from E. coli was developed using the X-ray crystal structure of the DapE from H. influenzae (PDB code:3IC1) as a template (Figure 3) (Rowsell et al. 1997; Eswar et al. 2003; Krissinel and Henrick 2004; Nocek et al. 2010). Sequence analysis of the target and template show that these two proteins are of similar length, share good sequence identity (~24%), and exhibit no significant sequence gaps. The ModWeb-based homology-building server was used to construct a structural homology model of the ArgE from E. coli. Comparison of the energy minimized ArgE homology model to the X-ray crystal structure of DapE, using the MathMaker in UCSF Chimera, reveals that the ArgE homology model displays the typical dimerization and catalytic domains of DapE with a Needleman-Wunsch (Needleman and Wunsch 1970) score of 490.6 and an average RMSD of 0.974 Å for the core atom pairs (Figure 3).Figure 3


Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli.

McGregor WC, Gillner DM, Swierczek SI, Liu D, Holz RC - Springerplus (2013)

Homology model of the [ZnZn(ArgE)] fromE. colibased on the X-ray structure of DapE. A) Overlay of the ArgE homology model (Green) and the X-ray crystal structure of DapE (Magenta). The two Zn(II) ions and the bridging water from ArgE are shown as spheres. B) Conserved active site residues for ArgE (Green) and DapE (Magenta). Nitrogen atoms are in blue, oxygen atoms are in red. The residues are labeled with single-letter amino acid codes with the labels for residues from DapE in parenthesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4320195&req=5

Fig3: Homology model of the [ZnZn(ArgE)] fromE. colibased on the X-ray structure of DapE. A) Overlay of the ArgE homology model (Green) and the X-ray crystal structure of DapE (Magenta). The two Zn(II) ions and the bridging water from ArgE are shown as spheres. B) Conserved active site residues for ArgE (Green) and DapE (Magenta). Nitrogen atoms are in blue, oxygen atoms are in red. The residues are labeled with single-letter amino acid codes with the labels for residues from DapE in parenthesis.
Mentions: A three-dimensional homological structure of the ArgE from E. coli was developed using the X-ray crystal structure of the DapE from H. influenzae (PDB code:3IC1) as a template (Figure 3) (Rowsell et al. 1997; Eswar et al. 2003; Krissinel and Henrick 2004; Nocek et al. 2010). Sequence analysis of the target and template show that these two proteins are of similar length, share good sequence identity (~24%), and exhibit no significant sequence gaps. The ModWeb-based homology-building server was used to construct a structural homology model of the ArgE from E. coli. Comparison of the energy minimized ArgE homology model to the X-ray crystal structure of DapE, using the MathMaker in UCSF Chimera, reveals that the ArgE homology model displays the typical dimerization and catalytic domains of DapE with a Needleman-Wunsch (Needleman and Wunsch 1970) score of 490.6 and an average RMSD of 0.974 Å for the core atom pairs (Figure 3).Figure 3

Bottom Line: The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble.Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE.Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM.

View Article: PubMed Central - PubMed

Affiliation: The Department of Applied Sciences and Mathematics, College of Technology and Innovation, Arizona State University, Mesa, AZ 85212 USA.

ABSTRACT
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

No MeSH data available.


Related in: MedlinePlus