RESUMEN

The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite.

Asunto(s)

DCMP Desaminasa/química , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxiuracil/química , Magnesio/química , Proteínas Protozoarias/química , Schistosoma mansoni/enzimología , Zinc/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cationes Bivalentes , Cristalografía por Rayos X , DCMP Desaminasa/genética , DCMP Desaminasa/metabolismo , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxiuracil/metabolismo , Expresión Génica , Cinética , Magnesio/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Zinc/metabolismo