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

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.

TRAF6 and p62 inhibit amyloid ß-induced neuronal death through p75 neurotrophin receptor.

Amyloid ß (Aß) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient's brain. Aß is known to bind p75 neurotrophin receptor (p75(NTR)) and mediates Aß-induced neuronal death. Recently, we showed that NGF leads to p75(NTR) polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aß stimulation impaired the p75(NTR) polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75(NTR) on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75(NTR) polyubiquitination upon Aß/NGF treatment. Aß significantly reduced NF-κB activity by attenuating the interaction of p75(NTR) with IKKß. p75(NTR) increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aß-mediated inhibition of p75(NTR) polyubiquitination and restored neuronal cell survival.

Structural characterization of Zn(II)-, Co(II)-, and Mn(II)-loaded forms of the argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.

The Zn, Co, and Mn K-edge extended X-ray absorption fine structure (EXAFS) spectra of the N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli, loaded with one or two equivalents of divalent metal ions (i.e., [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)], [Co(II)Co(II)(ArgE)], [Mn(II)_(ArgE)], and [Mn(II)Mn(II)(ArgE)]), were recorded. The Fourier transformed data (FT) for [Zn(II)_(ArgE)], [Zn(II)Zn(II)(ArgE)], [Co(II)_(ArgE)] and [Co(II)Co(II)(ArgE)] are dominated by a peak at 2.05Å, that can be fit assuming five or six light atom (N,O) scatterers. Inclusion of multiple-scattering contributions from the outer-shell atoms of a histidine-imidazole ring resulted in reasonable Debye-Waller factors for these contributions and a slight reduction in the goodness-of-fit value (f'). Furthermore, the data best fit a model that included a M-M vector at 3.3 and 3.4Å for Zn(II) and Co(II), respectively, suggesting the formation of a dinuclear site. Multiple scattering contributions from the outer-shell atoms of a histidine-imidazole rings are observed at ~3 and 4Å for Zn(II)- and Co(II)-loaded ArgE suggesting at least one histidine ligand at each metal binding site. Likewise, EXAFS data for Mn(II)-loaded ArgE are dominated by a peak at 2.19Å that was best fit assuming six light atom (N,O) scatterers. Due to poor signal to noise ratios for the Mn EXAFS spectra, no Mn-Mn vector could be modeled. Peak intensities for [M(II)_(ArgE)] vs. [M(II)M(II)(ArgE)] suggest the Zn(II), Co(II), and Mn(II) bind to ArgE in a cooperative manner. Since no structural data has been reported for any ArgE enzyme, the EXAFS data reported herein represent the first structural glimpse for ArgE enzymes. These data also provide a structural foundation for the future design of small molecules that function as inhibitors of ArgE and may potentially function as a new class of antibiotics.

Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.

The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-L-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k(cat) and K(m) values of 550 s(-1) and 0.8 mM, respectively, providing a catalytic efficiency (k(cat)/K(m)) of 6.9 x 10(5) M(-1) s(-1). The ArgE dissociation constant (K(d)) for Mn(II) was determined to be 0.18 microM, correlating well with a value obtained by isothermal titration calorimetry of 0.30 microM for the first metal binding event and 5.3 microM for the second. An Arrhenius plot of the NAO hydrolysis for Mn(II)-loaded ArgE was linear from 15 to 55 degrees C, suggesting the rate-limiting step does not change as a function of temperature over this range. The activation energy, determined from the slope of this plot, was 50.3 kJ mol(-1). Other thermodynamic parameters were DeltaG(double dagger) = 58.1 kJ mol(-1), DeltaH(double dagger) = 47.7 kJ mol(-1), and DeltaS(double dagger) = -34.5 J mol(-1) K(-1). Similarly, plots of lnK(m) versus 1/T were linear, suggesting substrate binding is controlled by a single step. The natural product, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]leucine (bestatin), was found to be a competitive inhibitor of ArgE with a K (i) value of 67 muM. Electron paramagnetic resonance (EPR) data recorded for both [Mn(II)_(ArgE)] and [Mn(II)Mn(II)(ArgE)] indicate that the two Mn(II) ions form a dinuclear site. Moreover, the EPR spectrum of [Mn(II)Mn(II)(ArgE)] in the presence of bestatin indicates that bestatin binds to ArgE but does not form a micro-alkoxide bridge between the two metal ions.

argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli contains a dinuclear metalloactive site.

The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).