title: Further insight into the inhibitory action of a LIM/double zinc-finger motif of an agmatinase-like protein.
abstract: Agmatine is a precursor for polyamine biosynthesis also associated to neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. It results from decarboxylation of l-arginine by arginine decarboxylase and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new protein which also hydrolyzes agmatine although its sequence greatly differs from all known agmatinases. This agmatinase-like protein (ALP) contains a LIM-like double Zn-finger domain close to its carboxyl terminus, whose removal results in a truncated variant with a 10-fold increased kcat, and a 3-fold decreased Km value for agmatine. Our proposal was that the LIM-domain functions as an autoinhibitory, regulatory entity for ALP. Results in this report provide additional support for the postulated inhibitory effect. The purified isolated LIM domain was shown to be competitively inhibitory to a truncated variant ALP (lacking the LIM-domain), but not to the wild-type species. The C453A variant was shown to be a Zn(2+)-free enzyme with kinetic parameters similar to those of the truncated-ALP. A molecular dynamic simulation of a modeled LIM-domain 3D structure showed that, as a consequence of C453A mutation, the coordination of the zinc ion is broken and the structure of the zinc finger is melted. The inhibitory action of the LIM/double Zinc-finger motif was associated to a significant conformational change, as detected by tryptophan fluorescence studies, but was not related to changes in the association of the enzyme with the catalytically essential Mn(2+).
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Jaime
authors.initials: J
authors.lastname: Cofre
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Paola
authors.initials: P
authors.lastname: Montes
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Vallejos
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: José
authors.initials: J
authors.lastname: Benítez
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: David
authors.initials: D
authors.lastname: García
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: José
authors.initials: J
authors.lastname: Martínez-Oyanedel
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
authors.forename: Nelson
authors.initials: N
authors.lastname: Carvajal
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile. Electronic address: auribe@udec.cl.
authors.forename: Elena
authors.initials: E
authors.lastname: Uribe
mesh_terms: D000595:Amino Acid Sequence; D056970:Co-Repressor Proteins / Q000737:chemistry*; D014644:Genetic Variation; D006801:Humans; D060588:LIM Domain Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D014512:Ureohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D056970:Co-Repressor Proteins; D060588:LIM Domain Proteins; C406239:LMCD1 protein, human; D014512:Ureohydrolases; C019625:agmatinase
keywords: Agmatinase-like protein; Agmatine; LIM-domain