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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

Surface rendering of the [ZnZn(ArgE)] molecule where the black box indicates the position of the di-nuclear metal center. Insert: Close-up of the dinuclear Zn(II) cluster showing that the active site is located in a long cleft across the catalytic domain. Zn(II) ions are shown in light blue spheres and the bridging water molecule is shown as a red sphere and labeled as “WAT”.
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Fig8: Surface rendering of the [ZnZn(ArgE)] molecule where the black box indicates the position of the di-nuclear metal center. Insert: Close-up of the dinuclear Zn(II) cluster showing that the active site is located in a long cleft across the catalytic domain. Zn(II) ions are shown in light blue spheres and the bridging water molecule is shown as a red sphere and labeled as “WAT”.

Mentions: Since no three-dimensional X-ray crystal structure has been reported for the ArgE form E. coli, a three-dimensional homological structure 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). The catalytic domain of ArgE contains the dinuclear Zn(II) site with a Zn-Zn distance of ~3.3 Å and within 4.0 Å of the Zn(II) binding sites of ArgE, and all of the amino acid residues are nearly identical to those observed in DapE confirming the assignment of H80 and H355 as active site ligands in ArgE (Figure 3) (Rowsell et al. 1997; Eswar et al. 2003; Krissinel and Henrick 2004). The active site in ArgE is located in a long and open cleft of the catalytic domain; the cleft is parallel to and in the vicinity of the “hinge” region between the catalytic domain and the dimerization domain (Figure 8). With such a location, conformational changes upon metal and/or substrate binding are likely and have been observed for DapE (Nocek et al. 2010). Both Zn(II) ions are solvent accessible and have four protein centered ligands including H80 and H355 as well as two terminal glutamate residues (E145 and E160) and a bridging aspartate residue (D122). A bridging water provides the fifth ligand for both metal ions, which forms a hydrogen bond to a second water molecule that is in turn hydrogen bonded to E144, a candidate for the general acid/base during catalysis (Davis et al. 2006). Therefore, our data supports the hypothesis that H355 is an active site Zn (II) ligand, and as this residue corresponds to H349 in DapE, which has been shown to be the second metal binding site, it is likely that H355 resides in the second metal binding site of ArgE.Figure 8


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)

Surface rendering of the [ZnZn(ArgE)] molecule where the black box indicates the position of the di-nuclear metal center. Insert: Close-up of the dinuclear Zn(II) cluster showing that the active site is located in a long cleft across the catalytic domain. Zn(II) ions are shown in light blue spheres and the bridging water molecule is shown as a red sphere and labeled as “WAT”.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig8: Surface rendering of the [ZnZn(ArgE)] molecule where the black box indicates the position of the di-nuclear metal center. Insert: Close-up of the dinuclear Zn(II) cluster showing that the active site is located in a long cleft across the catalytic domain. Zn(II) ions are shown in light blue spheres and the bridging water molecule is shown as a red sphere and labeled as “WAT”.
Mentions: Since no three-dimensional X-ray crystal structure has been reported for the ArgE form E. coli, a three-dimensional homological structure 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). The catalytic domain of ArgE contains the dinuclear Zn(II) site with a Zn-Zn distance of ~3.3 Å and within 4.0 Å of the Zn(II) binding sites of ArgE, and all of the amino acid residues are nearly identical to those observed in DapE confirming the assignment of H80 and H355 as active site ligands in ArgE (Figure 3) (Rowsell et al. 1997; Eswar et al. 2003; Krissinel and Henrick 2004). The active site in ArgE is located in a long and open cleft of the catalytic domain; the cleft is parallel to and in the vicinity of the “hinge” region between the catalytic domain and the dimerization domain (Figure 8). With such a location, conformational changes upon metal and/or substrate binding are likely and have been observed for DapE (Nocek et al. 2010). Both Zn(II) ions are solvent accessible and have four protein centered ligands including H80 and H355 as well as two terminal glutamate residues (E145 and E160) and a bridging aspartate residue (D122). A bridging water provides the fifth ligand for both metal ions, which forms a hydrogen bond to a second water molecule that is in turn hydrogen bonded to E144, a candidate for the general acid/base during catalysis (Davis et al. 2006). Therefore, our data supports the hypothesis that H355 is an active site Zn (II) ligand, and as this residue corresponds to H349 in DapE, which has been shown to be the second metal binding site, it is likely that H355 resides in the second metal binding site of ArgE.Figure 8

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