Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB |
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Authors: | Alicia Cabezas,Iralis Ló pez-Villamizar,Marí a Jesú s Costas,José Carlos Cameselle,Joã o Meireles Ribeiro |
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Affiliation: | 1.Grupo de Enzimología, Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina y Ciencias de la Salud, Universidad de Extremadura, 06006 Badajoz, Spain; (A.C.); (I.L.-V.); (M.J.C.); (J.C.C.);2.Manlab, Diagnóstico Bioquímico y Genómico, Calle Marcelo Torcuato de Alvear 2263, 1122 Ciudad de Buenos Aires, Argentina |
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Abstract: | The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains. |
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Keywords: | chimeragenesis, protein domain, substrate-binding site, catalytic site, substrate specificity, 5′ -nucleotidase, 3′ -nucleotidase, phosphodiesterase, phosphoanhydride hydrolase |
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