Structural Insights into the Catalytic Mechanism of Sphingomyelinases D and Evolutionary Relationship to Glycerophosphodiester Phosphodiesterases
Mário T. Murakamia; Matheus Freitas Fernandes-Pedrosab; Sonia A. de Andradeb; Azat Gabdoulkhakovc; Christian Betzeld; Denise V. Tambourgib,e; Raghuvir K. Arnia,e.
aDepartment of Physics, IBILCE/UNESP, São Jose do Rio Preto-SP, Brazil; bImmunochemistry Laboratory, Butantan Institute, Sao Paulo-SP, Brazil; cInstitute for Protein Research RAS, Moscow, Russia; dDivision of Biochemistry and Molecular Biology, Hamburg University, Germany; eCenter for Applied Toxinology, Butantan Institute, Sao Paulo-SP, Brazil.
Spider venom sphingomyelinases D catalyze the hydrolysis of sphingomyelin via an Mg2+ ion-dependent acid–base catalytic mechanism which involves two histidines. In the crystal structure of the sulfate free enzyme determined at 1.85 Å resolution, the metal ion is tetrahedrally coordinated instead of the trigonal–bipyramidal coordination observed in the sulfate bound form. The observed hyperpolarized state of His47 requires a revision of the previously suggested catalytic mechanism. Molecular modeling indicates that the fundamental structural features important for catalysis are fully conserved in both classes of SMases D and that the Class II SMases D contain an additional intra-chain disulphide bridge (Cys53–Cys201). Structural analysis suggests that the highly homologous enzyme from Loxosceles bonetti is unable to hydrolyze sphingomyelin due to the 95Gly à Asn and 134Pro à Glu mutations that modify the local charge and hydrophobicity of the interfacial face. Structural and sequence comparisons confirm the evolutionary relationship between sphingomyelinases D and the glicerophosphodiester phosphoesterases which utilize a similar catalytic mechanism.
This research was supported financially by grants from FAPESP / SMOLBNet / CEPID, CNPq and CAPES/DAAD.
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