Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family.
A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn(2+); K(m) and k(cat) values were 2.5+/-0.2 mM and 0.8+/-0.2 s(-1), respectively. The product putrescine was a linear competitive inhibitor (K(i)=5+/-0.5 mM). The substrate specificity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k(cat)/K(m) (320 M(-1)s(-1)), the recombinant protein was about 290-fold less efficient than the bacterial enzyme. The deduced amino sequence revealed great differences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.
Uribe
Elena
E
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
Salas
Mónica
M
Enríquez
Silvia
S
Orellana
María Soledad
MS
Carvajal
Nelson
N
D000595:Amino Acid Sequence; D000818:Animals; D001119:Arginase / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D051381:Rats; D014512:Ureohydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D018076:DNA, Complementary; D014512:Ureohydrolases; D001119:Arginase; C019625:agmatinase