Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Binding of azide and thiocyanate ligands to copper(II) model complexes

Summary Binding of azide to a series of copper(II) complexes has been investigated by absorption, CD and EPR spectroscopy. Axial binding of azide to Cu(II) can be differentiated from equatorial binding through the lower intensity and lack of optical activity of the LMCT band. The affinity of azide for Cu(II) increases with the overall positive charge of the complex. The preliminary data on thiocyanate binding to Cu(II) seem to agree with the trends observed for the corresponding azide adducts.     

Cobalt-substituted derivatives ofCarcinus hemocyanin

Summary Four derivatives ofCarcinus maenas hemocyanin containing Co(II) in the active site have been prepared under different experimental conditions. Two of them contain one Co(II) ion/active site and most probably represent isomeric forms containing Co(II) either in the fast-reacting or in the slow-reacting position within the active site. A third derivative contains two Co(II) ions active site, which reproduces the metal/protein stoichiometry of native hemocyanin. The fourth derivative is a metal hybrid form containing one Cu(I) ion and one Co(II) ion/active site. The derivatives have been characterized by absorption, circular dichroic and fluorescence spectroscopies. The results indicate that in all derivatives the metal is bound with a low coordination number, in agreement with the presence of three histidine residues/copper ion in the native protein. The two alternative metal-binding positions have different structures as shown by the different spectroscopic properties of the bound Co(II) ions. A marked hyperchromic effect on the optical absorption of Co(II) is observed as a result of the presence of a metal ion in the neighbouring metal-binding position in the active site.     

The aromatic circular dichroism spectrum as a probe for conformational changes in the active site environment of hemocyanins.

The binding of various ligand molecules to the binuclear Cu(I) site of deoxy-hemocyanin has been investigated through the changes produced in the aromatic region of the circular dichroism spectrum of the protein, where a cluster of tryptophan residues located in the vicinity of copper site undergo conformational reorientations in the presence of exogenous ligands coordinated to the metal. In agreement with expectations, the binuclear site of arthropod hemocyanin is severely hindered to the access of exogenous ligands except for very small molecules like CO, O2 or CN- while for mollusc proteins ligands such as thiourea and 2-mercaptoethanol bind easily to the Cu(I) sites. However, the access of the ligand becomes progressively hindered and eventually prevented as the size of substituents on the ligand increases.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.