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J. Biol. Chem., Vol. 280, Issue 14, 13658-13664, April 8, 2005

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Structural Basis for Metal Ion Coordination and the Catalytic Mechanism of Sphingomyelinases D^*

Mário T. Murakami, Matheus F. Fernandes-Pedrosa¶, Denise V. Tambourgi¶||, and Raghuvir K. Arni**

From the Department of Physics, Instituto de Biociências, Letras e Ciências Exatas/Universidade Estadual Paulista, São José do Rio Preto, SP 15054-000, Brazil, and the ^¶Immunochemistry Laboratory, Butantan Institute, São Paulo, SP 05503-900, Brazil

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-Å resolution using the "quick cryo-soaking" technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (/)₈ barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg²⁺ ion, which is coordinated by Glu³², Asp³⁴, Asp⁹¹, and solvent molecules. In the proposed acid base catalytic mechanism, His¹² and His⁴⁷ play key roles and are supported by a network of hydrogen bonds between Asp³⁴, Asp⁵², Trp²³⁰, Asp²³³, and Asn²⁵².

Received for publication, November 3, 2004 , and in revised form, January 10, 2005.

^* * This research was supported by grants from FAPESP (SMOLBNet), CNPq and CAPES/DAAD (to R. K. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1XX1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Recipients of FAPESP doctoral fellowships.

^|| Supported by FAPESP and The Welcome Trust.

^** To whom correspondence should be addressed: Dept. of Physics, IBILCE/UNESP, Rua Cristovão Colombo 2265, São José do Rio Preto, SP 15054-000, Brazil. Tel.: 55-17-2212460; Fax: 55-17-2212247; E-mail: arni{at}df.ibilce.unesp.br.

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