A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning.

A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the N-terminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1-1 (GST P1-1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35 degrees C, glutathione (GSH) binding to GST P1-1 displays positive cooperativity, whereas negative cooperativity occurs below 25 degrees C. This binding mechanism minimizes changes of GSH affinity for GST P1-1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1-1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding

Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli

An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaperonins GroEL and GroES, a R action of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may be obtained by genetic engineering technology. (C) 1995 Academic Press, Inc

Nitrosylation of human glutathione transferase P1-1 with dinitrosyl diglutathionyl iron complex in vitro and in vivo.

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1