The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation.

The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn(2+) ions. Mutation of conserved residues that coordinate Mn(2+) ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle.

The Pih1-Tah1 cochaperone complex inhibits Hsp90 molecular chaperone ATPase activity.

Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.

Inactivation of human arylamine N-acetyltransferase 1 by hydrogen peroxide and peroxynitrite.

Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for the acetylation of many arylamine and heterocyclic amines. They therefore play an important role in the detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is present mainly in the liver and intestine, whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and/or pathological susceptibility. Evidence now shows that redox conditions may also contribute to overall NAT activity. This chapter summarizes current knowledge on human NAT1 regulation by reactive oxygen and nitrogen species.

Skeletal muscles express the xenobiotic-metabolizing enzyme arylamine N-acetyltransferase.

The human arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are enzymes responsible for the acetylation of many arylamines and hydrazines, thereby playing an important role in both detoxification and activation of many drugs and carcinogens. Both enzymes show polymorphisms but exhibit key differences in substrate selectivity and tissue expression. In the present study, reverse transcriptase-PCR, Western blotting, and immunohistochemistry were used to investigate the expression of the NATs in human skeletal muscle. Despite the presence of its mRNA, NAT2 enzyme level was below the limit of detection. In contrast, both NAT1 mRNA and enzyme were readily detected in fetal, newborn, and adult muscles. In addition, punctate cytoplasmic and perinuclear NAT1 immunostaining was observed in all tissue sections, the staining being more intense in the fetal tissue. High expression of NAT1 enzyme in fetal muscle was also suggested by Western blotting. Because skeletal muscle accounts for a large proportion of body mass, muscle NAT1 expression may contribute significantly to the total activity in the body. These results further support the involvement of skeletal muscle in the metabolism of xenobiotics.

The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1.

The arylamine N-acetyltransferases are important xenobiotic-metabolizing enzymes that catalyze an acetyl group transfer from acetylCoA to arylamine substrates. NAT enzymes possess an active-site loop (the active-site P-loop) involved in substrate binding and selectivity. The Gly/Ala residue present at the start of the active-site P-loop, although conserved in all NAT enzymes, is not involved in the catalytic mechanism or substrate binding. Here we show that a small amino acid (such as Gly or Ala) at this position is important not only for maintaining the functions of the active-site P-loop but, more surprisingly, also important for maintaining the overall structural integrity of NAT enzymes. Our data thus suggest that in addition to its role in substrate binding and selectivity, the active-site P-loop could play a wider structural role in NAT enzymes.

Peroxynitrite irreversibly inactivates the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) in human breast cancer cells: a cellular and mechanistic study.

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols.

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.

Redox regulation of the human xenobiotic metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1). Reversible inactivation by hydrogen peroxide.

Oxidative stress is increasingly recognized as a key mechanism in the biotransformation and/or toxicity of many xenobiotics. Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic ubiquitous phase II xenobiotic metabolizing enzyme that catalyzes the biotransformation of primary aromatic amine or hydrazine drugs and carcinogens. Functional and structural studies have shown that NAT1 catalytic activity is based on a cysteine protease-like catalytic triad, containing a reactive cysteine residue. Reactive protein cysteine residues are highly susceptible to oxidation by hydrogen peroxide (H2O2) generated within the cell. We, therefore, investigated whether human NAT1 activity was regulated by this cellular oxidant. Using purified recombinant NAT1, we show here that NAT1 is rapidly (kinact = 420 m-1.min-1) inactivated by physiological concentrations of H2O2. Reducing agents, such as reduced glutathione (GSH), reverse the H2O2-dependent inactivation of NAT1. Kinetic analysis and protection experiments with acetyl-CoA, the physiological acetyl-donor substrate of the enzyme, suggested that the H2O2-dependent inactivation reaction targets the active-site cysteine residue. Finally, we show that the reversible inactivation of NAT1 by H2O2 is due to the formation of a stable sulfenic acid group at the active-site cysteine. Our results suggest that, in addition to known genetically controlled interindividual variations in NAT1 activity, oxidative stress and cellular redox status may also regulate NAT1 activity. This may have important consequences with regard to drug biotransformation and cancer risk.