2 research outputs found
Importance of Val567 on heme environment and substrate recognition of neuronal nitric oxide synthase
Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the activeāsite residue Val567 (numbering for rat neuronal NOS, nNOS). NOSālike proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NOābinding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UVāvisible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the hemeāFeIIāCO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of hemeābound gaseous ligands, and potential NO production
Structural basis for enzymatic excision of N(1)-methyladenine and N(3)-methylcytosine from DNA
N(1)-methyladenine (m(1)A) and N(3)-methylcytosine (m(3)C) are major toxic and mutagenic lesions induced by alkylation in single-stranded DNA. In bacteria and mammals, m(1)A and m(3)C were recently shown to be repaired by AlkB-mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m(1)A and m(3)C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N(6)-ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several site-directed mutants, forms a molecular rationalization for the newly discovered base excision activity