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 (Kd) 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.
Highlights
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, only the H355A enzyme was found to be soluble
Encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) (Nocek et al 2010), the N-acetyl-L-citrulline deacetylase (ACD) from Xanthomonas campestris (Shi et al 2007), and the carboxypeptidase from Pseudomonas sp strain-RS-16 (CPG2) (Rowsell et al 1997), the residues that function as ligands in the dinuclear active site of Aeromonas proteolytica (AAP), dapE-encoded N-succinyl-L (DapE), ACD, and carboxypeptidase from Pseudomonas sp. strain-RS-16 (CPG2) are strictly conserved in ArgE (Figure 2) (Born et al 1998; Chevrier et al 1994; Rowsell et al 1997)
Protein production Of the four ArgE mutant plasmids produced (H355A, H355K, H80A and H80K) and transformed into E. coli BL21 StarTM cells, only ArgE H355A was produced as a soluble enzyme
Summary
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, only the H355A enzyme was found to be soluble. + H2N encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) (Nocek et al 2010), the N-acetyl-L-citrulline deacetylase (ACD) from Xanthomonas campestris (Shi et al 2007), and the carboxypeptidase from Pseudomonas sp strain-RS-16 (CPG2) (Rowsell et al 1997), the residues that function as ligands in the dinuclear active site of AAP, DapE, ACD, and CPG2 are strictly conserved in ArgE (Figure 2) (Born et al 1998; Chevrier et al 1994; Rowsell et al 1997). In an effort to provide insight into the structural properties of each divalent metal ion in the active site of the ArgE form E. coli, we have prepared and purified the H355A, H355K, H80A, and H80K ArgE mutant enzymes
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