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Originally published In Press as doi:10.1074/jbc.M412437200 on January 14, 2005

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{ddagger}§, Matheus F. Fernandes-Pedrosa§, Denise V. Tambourgi¶||, and Raghuvir K. Arni{ddagger}**

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({alpha}/{beta})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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Envenomation by arachnids of the genus Loxosceles (brown spider), endemic to temperate and tropical regions of the Americas, Africa, and Europe, leads to local dermonecrosis and also to serious systemic toxicity. Three principal Loxosceles species of medical importance are encountered in Brazil (Loxosceles laeta, Loxosceles intermedia, Loxosceles gaucho), and more than 2,000 cases of envenomation by L. intermedia alone are reported each year. In the United States, six Loxosceles species (including Loxosceles reclusa, brown recluse) are responsible for numerous incidents (1). L. laeta, possibly the most toxic and dangerous of all the species, is widely distributed and is encountered as far north as Canada (2, 3) and is endemic primarily in South and Central America.

The site of envenomation, which initially causes only minor discomfort, begins as an expanding area of erythema and edema. A centrally located necrotic ulcer often forms 8–24 h after envenomation (4, 5). Extensive tissue destruction follows, with the ulcer taking many months to heal, and in extreme cases requires debridement or skin grafting. The lesions are remarkable considering that Loxosceles spiders inject only a few tenths of a microliter of venom containing no more than 30 µg of protein.

Mild systemic effects induced by envenomation, such as fever, malaise, pruritus, and exanthema are common, whereas intravascular hemolysis and coagulation, sometimes accompanied by thrombocytopenia and renal failure, occur in ~16% of the victims (1, 6–11). Although systemic loxoscelism is less common than the cutaneous form, it is the main cause of death associated with Loxosceles envenomation. Most of the deaths occur in children and are related to the South American species L. laeta (1). Due to our limited understanding of the venom's mechanism of action, effective treatment is currently not available.

The recombinant sphingomyelinases (SMases)1 D of L. laeta and L. intermedia retain all the local and systemic effects observed in the whole venom, inducing dermonecrosis in rabbits and rendering human erythrocytes susceptible to lysis by complement (12–15). In a mouse model of Loxosceles envenomation, they also induce intravascular hemolysis and provoke a cytokine response, which resembles that observed in endotoxic shock (16). SMases facilitate activation of the alternative pathway of complement on human erythrocytes by removal of glycophorins as a consequence of the activation of an endogenous metalloproteinase (17) and activation of the classical pathway of complement, possibly by disruption of the membrane asymmetry (18). SMases D are not encountered elsewhere in the animal kingdom; however, a similar enzyme is produced as an exotoxin by some pathogenic bacteria, notably Corynebacterium pseudotuberculosis, Corynebacterium ulcerans, and Arcanobacterium (formerly Corynebacterium) hemolyticum (19–21). C. pseudotuberculosis causes lymphadenitis in animals and is also pathogenic to humans, whereas C. ulcerans and A. hemolyticum are pathogens of pharyngitis and other human infections (22). The SMase D from C. pseudotuberculosis, also named sphingomyelin (SM)-specific phospholipase D (PLD), is an essential virulence determinant that contributes to the persistence and spread of the bacteria within the host (23).

The Loxosceles and bacterial SMases D possess similar molecular masses (31–35 kDa) but share only limited sequence homology (13, 24). In model systems, the Loxosceles and C. pseudotuberculosis enzymes provoke similar pathophysiological effects, including platelet aggregation, endothelial hyperpermeability, complement-dependent hemolysis, and neutrophil recruitment (13–15, 25–28).

Of the four major phospholipids present in the outer leaflet of the mammalian plasma membranes, only sphingomyelin is hydrolyzed by bacterial PLD and spider toxins, resulting in the formation of ceramide-1-phosphate (Cer-1-P or N-acyl-sphingosine-1-phosphate) (13, 25, 26).

In the presence of Mg2+, spider and bacterial SMases D catalyze the release of choline from lysophosphatidylcholine but not from phosphatidylcholine (24). Plasma lysophosphatidylcholine is tightly bound to albumin, and removal of its choline headgroup yields lysophosphatidic acid, a potent lipid mediator with numerous biological activities in many different cell types (29, 30). The crystal structure of SMase I (sphingomyelin phosphodiesterase; E.C. 3.1.4.12 [EC] ), one of the sphingomyelinase D isoforms from L. laeta venom (determined at 1.75 Å), provides a structural basis for understanding the role of the metal ion binding and the acid-base catalytic mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sphingomyelinase Expression—L. laeta SMase I (GenBankTM accession number AY093599 [GenBank] ) was expressed in Escherichia coli strain BL21 (DE3) as a fusion protein composed of the mature SMase with an N-terminal extension containing a His6 tag (15). Recombinant SMase I was purified from the soluble fraction of cell lysates on a Ni(II)-chelating-Sepharose Fast Flow column (Amersham Biosciences). Recombinant protein was eluted (elution buffer: 100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.8 M imidazole) at >95% purity and dialyzed against phosphate-buffered saline, pH 7.2 (10 mM sodium phosphate, 150 mM NaCl). Dynamic light-scattering experiments carried out in the above buffer at 293 K (DynaPro 801-Protein Solutions) indicated that the protein was monomeric in solution.

Crystallization, Heavy Atom Derivative, and Data Collection—Initial crystals of L. laeta SMase I were obtained by the hanging drop vapor diffusion method in which 2-µl drops containing 1 µl of the protein solution (5 mg ml–1 in 25 mM Hepes, pH 7.5) were equilibrated against a reservoir solution containing 8 mM Hepes and 0.9 M trisodium citrate (pH 7.5) (31). Subsequently, crystals obtained from 2.7 M ammonium sulfate (pH 5.6) were easier to reproduce and were used for structure determination. Cryo-conditions included the addition of 20% (w/v) glycerol to the reservoir solution. The native crystals belong to the space group P65 with cell parameters of a = b = 139.82 Å and c = 113.46 Å, and the asymmetric unit contains four molecules with a solvent content of 52% (VM = 2.6 Å3 Da–1). The iodine derivative for the "quick cryo-soaking" method was prepared by soaking a single crystal in the cryo-solution, which additionally contained 0.5 M iodine chloride, for 20 min. Diffraction intensities for the native crystal were measured by a MARCCD detector at the protein crystallography beam line at the Brazilian National Synchrotron Light Source (LNLS, Campinas, Brazil), and the derivative diffraction data were collected using x-rays generated by a Rigaku RU300 rotating anode source equipped with Osmic confocal mirrors. Diffraction intensities were measured using a MAR 345 imaging plate detector. Data were scaled and reduced using DENZO/SCALEPACK (32); the data collection and processing statistics are presented in Table I.


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  		TABLE I
Data collection and refinement statistics of SMase I

 
Structure Determination and Refinement—The quick cryo-soaking method (33, 34) was used for derivatization and phasing. The structure was determined at 1.95 Å using the single isomorphous replacement anomalous dispersion method, and the anomalous differences were used to locate 41 iodine sites with the SHELXD program (35) by integrated-direct and Patterson methods. These heavy atom positions were used without further refinement to estimate phases that were subsequently extended to 1.75-Å resolution applying the sphere of influence algorithm as incorporated in the SHELXE program (35), and the electron density was improved by solvent flattening with SOLOMON (36). The deduced amino acid sequence based on the L. laeta SMase I gene (GenBankTM accession number AAM21154 [GenBank] was utilized for automatic model building into the 1.75-Å resolution electron density map with the ARP/wARP program (37), which was able to trace 97% of the molecule. The refinement was initiated at a 2.0-Å resolution with an R-factor of 22.2% (Rfree = 26.4%). Initial cycles of refinement involved translation, libration, and screw rotation, a restrained and overall B-factor refinement that was carried out by REFMAC5 (38) with the inclusion of non-crystallographic restraints. After each cycle of refinement, the model was inspected and manually adjusted to correspond to the computed {sigma}A-weighted (2Fo – Fc)- and (Fo – Fc)-type electron density maps using the program TURBO FRODO (Biographics, Marseille, France). In the later cycles, the non-crystallographic restraints were relaxed, and individual isotropic B-factors were refined. Solvent water molecules were added manually at the position of positive peaks (>3{sigma}) in the difference Fourier maps, taking into consideration hydrogen bonding potential. In the final cycles of refinement, additional density was observed in the difference electron density maps, which was attributed to the presence of Mg2+, sulfate ions, and Hepes molecules.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The refinement of the structure of SMase I from L. laeta converged to a crystallographic residual of 18.6% (Rfree = 22.5% for 5% of the data) for all data between 30.0 and 1.75 Å (no {sigma} or intensity cutoff; 99.5% data completeness). The residual electron density that was observed was attributed to the presence of a Mg2+ ion based on the temperature factor (B-factor = 7.5 Å2) and coordination (Figs. 1 and 2A). Because the concentration of the phosphate in the dialysis buffer (10 mM) was lower than the sulfate concentration (2.6 M) used in crystallization, the tetrahedrally shaped residual density observed in close proximity to the Mg2+ binding site was considered to represent an SO4– ion (Fig. 2A). Although hydrolytic activity is Mg2+-dependent and this site is surrounded by highly conserved amino acids, it is considered to represent the active site, and the SO4– ion likely mimics the binding of the phosphate group of the substrate.



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  		FIG. 1.
Stereo ribbon representation of the structure of L. laeta SMase I viewed along the axis of the ({alpha}/{beta})8 barrel. The N and C termini are labeled, and the functionally important His12, Glu32, Asp34, His47, Asp91, and the Mg2+ ion (green sphere) are included. The catalytic loop B (blue), the variable loop E (green), and the flexible loop F (red) are indicated. The {beta}-strands and {alpha}-helices are labeled as in Fig. 3. Figs. 1, 2A, 4, and 6 were generated using Pymol (DeLano, www.pymol.org).

 



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  		FIG. 2.
A, stereo view of the amino acids and hydrogen bonding in the catalytic and metal ion binding (green sphere) sites; the electron density (blue) in the 2Fo – Fc map is contoured at 2.0{sigma}. B, schematic representation of the principal hydrogen bonds to the sulfate and metal ion.

 
The refined model of SMase I contains 1140 amino acid residues, 4 Mg2+ ions, 23 sulfate ions, 3 Hepes, and 1018 solvent water molecules. An analysis of the stereochemistry (PROCHECK) (39) of the final model indicates that the main-chain dihedral angles for all residues are located in the permitted regions of the Ramachandran diagram and that the root mean square deviations from ideal values are distributed within the expected ranges for a well refined structure (Table I).

The fundamental structural unit of SMase I is formed by a distorted triose phosphate isomerase or ({alpha}/{beta})8 barrel (surface area = 11,254 Å2) with the insertion of additional {beta}-strands and {alpha}-helices (Figs. 1 and 3). The N and C termini flank one side of the barrel, and the N terminus leads directly to the first {beta}-strand, whereas the C terminus contains a short helix (8') and a {beta}-strand (H') and caps the torus of the barrel (Figs. 1 and 3). The asymmetric unit contains four molecules and can be considered to be made up of two dimers. The monomers forming the dimer are related by a 2-fold axis of rotation perpendicular to the barrel (Fig. 4) and results in 3402 Å2 (or 33%) of the surface area of each monomer being buried. Dimerization is likely an artifact of the crystal packing interactions, because the dynamic light-scattering experiments indicate that the protein is monomeric in solution.



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  		FIG. 3.
Topology schematic of L. laeta SMase I. The {beta}-strands (arrows) and {alpha}-helices (cylinders) forming the ({alpha}/{beta})8 barrel are labeled A–H and 1–8 respectively. The {beta}-strands and {alpha}-helices not belonging to the core are primed. The positions of the catalytic loop B (blue), variable loop E (green), flexible loop F (red), and the disulfide bridge (S–S) are indicated. The approximate relative positions of the amino acids involved in catalysis and Mg2+ ion binding are indicated.

 



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  		FIG. 4.
Ribbon representation of the dimer. The amino acids His12, Glu32, Asp34, His47, and Asp91 are represented by balls and sticks, and the Mg2+ ion is represented as a green sphere. Residues in the catalytic (loop B), variable (loop E), and the flexible (loop F) loops are blue, green, and red, respectively. The N and C termini are labeled and are brown.

 
The opposite face of the barrel is surrounded by a ring of negatively charged amino acids and hydrophobic loops (Fig. 1). The active site pocket contains His12, Glu32, Asp34, Asp91, His47, Asp52, Trp230, Asp233, and Asn252, which are fully conserved in the Loxosceles species SMases D isoforms (Fig. 5). Mutagenesis studies of Mg2+-dependent neutral SMase (40) and the crystal structure of phospholipase D (41) indicate the involvement of two histidine residues that are in close proximity to the metal ion binding site in the acid-base catalytic mechanism. Based on the structural results, His12 and His47 of SMase D have been identified as the key residues for catalysis and are assisted by a hydrogen bond network that involves Asp52, Asn252, and Asp233. The metal ion is coordinated by Glu32, Asp34, Asp91, and solvent molecules.



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  		FIG. 5.
Structure-based multiple sequence alignments of the SMases D isoforms performed using three-dimensional Coffee 2004 (49). L. laeta: SMase I, H10, and H13 (GenBankTM accession numbers AY093599 [GenBank] , AY093600 [GenBank] and AY093601 [GenBank] , respectively) (15); L. intermedia: P1 and P2 (accession numbers AY304471 [GenBank] and AY304472 [GenBank] , respectively) (14); L. reclusa: Lr1 and Lr2 (accession numbers AY559846 [GenBank] and AY559847 [GenBank] , respectively) (19); Loxosceles boneti: Lb1 and Lb3 (accession numbers AY559844 [GenBank] and AY559845 [GenBank] , respectively) (50) and C. pseudotuberculosis: PLD (accession number L16586 [GenBank] ) (21). Residues involved in catalysis or metal ion coordination are shaded gray. Amino acids in loops B, E, and F are boxed. Sequence numbers indicated are for L. laeta SMase I.

 
His12 is located at the tip of the first {beta}-strand (strand A, Figs. 1 and 3), and N{epsilon}2 forms a hydrogen bond to O2 of the bound sulfate ion. His12N{delta}1 is hydrogen bonded to Asn252O{delta}1 and Asn252N{delta}2 is further hydrogen bonded to Asp52O{delta}2, and Asp233O{delta}2 (Fig. 2).

The catalytic loop (loop B, residues 46–60) is stabilized at the tip by a disulfide bridge (Cys51-Cys57, Figs. 3 and 6), which is a conserved feature of all spider SMases D (Fig. 5), and His47 is located at the base of the loop. Multiple hydrogen bonds are formed between Arg55NH1 and the main-chain carbonyl oxygen of His12 and Arg55NH2 and Met13O (Fig. 6), and additionally Arg55NH1 also bonds Asp252O{delta}1. A structural sulfate ion binds via O1 to Arg55N{epsilon} and the main chain amide nitrogen of Asp56. O4 of the sulfate ion binds two solvent molecules, both of which are hydrogen bonded to Arg59NH1. Arg59N{epsilon} binds a molecule of Hepes, the latter interacting with the indole of Trp60 located at the base of the catalytic loop (loop B) on the external surface of the barrel. This network of bonds ensures the orientation of the catalytic loop in relation to the active site. His47N{epsilon}2 is hydrogen bonded to O1 and O3 of the sulfate ion located in the catalytic site, and His47N{delta}1 interacts with the carbonyl oxygen atom of Gly48 (Fig. 2B).



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  		FIG. 6.
Network of hydrogen bonds (dashed red lines) participating in the stabilization of the catalytic loop (loop B). The structural SO4– ion, disulfide bridge (yellow bond), and solvent molecules (red spheres) are also included.

 
The bound Mg2+ ion (Fig. 2) is octahedrally coordinated (mean Mg2+–O distance of 1.98 Å), equatorially by carboxylate oxygens from the side chains of Glu32, Asp34, and two water molecules and apically by the side-chain carboxylate oxygen atoms of Asp91 and a water molecule, which is additionally hydrogen bonded to Glu32O{epsilon}1. The sulfate ion is coordinated by three solvent molecules, two of which also coordinate the Mg2+ ion, His12N{epsilon}2, His47N{epsilon}2, and Trp230N{epsilon}1. Trp230 is located in loop G (Fig. 1), which is structurally adjacent to the flexible loop (loop F) and is strictly conserved in bacterial and spider SMases D. The indole ring is partially disordered in three of the four molecules in the asymmetric unit, is aligned along the axis of the barrel, and could be involved in stabilizing the choline headgroup of the substrate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The branching pathways of sphingolipid metabolism mediate either apoptotic or mitogenic responses, depending on the cell type and the nature of the stimulus (42). Events involving SM metabolites include proliferation, differentiation, and growth arrest, as well as the induction of apoptosis.

Loxosceles spiders and Corynebacteria SMases D catalyze the hydrolysis of sphingomyelin in a Mg2+-dependent manner, with the concerted action of two histidines producing ceramide 1-phosphate and choline, and also display intrinsic lysophospholipase D activity toward lysophosphatidylcholine producing lysophosphatidic acid, a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses. However, sequence alignments indicate that SMases D lack the conserved HKD sequence motif characteristic of the PLD superfamily (43), indicating different catalytic site architectures.

The Mg2+ binding site is strictly conserved in spider and C. pseudotuberculosis SMases D (Fig. 5), and enzymatic activity is absolutely dependent on Mg2+. In the crystal structure, the Mg2+ ion is octahedrally coordinated by the carboxyl oxygens of Glu32, Asp34, Asp91, and three solvent molecules. The Mg2+ ion positions the two equatorial solvent molecules, which in turn could orient the phosphate group of the substrate or could participate in the stabilization of the reaction intermediate. Trp230, which is fully conserved in SMases D, could also play a role in orienting the phosphate moiety, permitting nucleophilic attack, and also in stabilizing the transition state. Lys93, located in the catalytic pocket, is also highly conserved and may play a crucial role in balancing the charge during catalysis or in orienting the bound substrate.

Although mammalian desoxyribonuclease I and bacterial SMases C share <10% sequence identity, the two enzymes are considered to be evolutionarily related, and the structure of desoxyribonuclease I (44, 45) (Protein Data Bank code 3DNI [PDB] ) has been used as a template to model the structure of Bacillus cereus SMase C (46). Based on the results of modeling and site-directed mutagenesis, an acid-base mechanism has been suggested for bacterial and mammalian Mg2+-dependent neutral sphingomyelinases (40, 46), where His296 activates a neighboring water molecule, which in turn attacks the scissile phosphodiester bond of SM. The electron then transfers to the general acid (His151) through the penta-covalent intermediate, resulting in the release of ceramide from SM. The intermediate is then stabilized by Mg2+ liganded by Glu53, Asp195, and Asp295, which suggests that the latter play dual roles by maintaining the appropriate pKa and relative orientation of His151 and His296. Mutation of either His136 or His272 in rat neutral sphingomyelinase or Asp195 and His296 in B. cereus (40) sphingomyelinase entirely abolished hydrolytic activity. The His151->Ala and His151->Gln mutants of the B. cereus enzyme retained partial activity (40).

We are now able to infer the catalytic mechanism of SMase D, which is based on the direct nucleophilic attack of water in a fashion analogous to the mechanisms proposed for desoxyribonuclease I (44, 45, 47), PLD (41–48), and for B. cereus SMase C (46). In this model (Fig. 7), the concerted action of two histidines (His12 and His47) is required for catalysis. His12 functions as the nucleophile that initiates the attack on the scissile phosphodiester bond of the SM substrate. This is followed by the formation of a short-lived penta-coordinated covalent intermediate, which is subsequently destabilized by the donation of a hydrogen atom by His47 to produce choline. The resulting tetrahedral reaction intermediate is stabilized by a covalent bond formed to His12N{epsilon}2. Because His47 donates a proton to the group first to leave, it is now able to (partially) deprotonate a nearby water molecule that initiates a nucleophilic attack on the stable covalent histidine intermediate, thereby resulting in the formation of the second product, ceramide 1-phosphate, which also proceeds via the formation of a short-lived, penta-coordinated phosphorus intermediate, culminating in a second inversion of the configuration of the phosphorus atom and a return to the initial state.



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  		FIG. 7.
The proposed mechanism for the catalytic hydrolysis of the sphingomyelin substrate by SMase I, His12, and His47 participate in the reaction as the acid and base. R and R' represent ceramide 1-phosphate and choline, respectively. The figure was generated using ChemSketch (www.acdlabs.com).

 
In this proposed model, His12 functions as the nucleophile, which is assisted by a network of hydrogen bonds formed to the carboxylate oxygen of Asn252, which in turn is hydrogen bonded through N{delta}2 to Asp52O{delta}2 and Asp233O{delta}2. Additionally, His12N{delta}1 is also hydrogen bonded to Asp52O{delta}2. His47N{delta}1 is hydrogen bonded to the carbonyl oxygen atom of Gly48 (distance = 2.7 Å) and thus cannot serve as a proton donor in this orientation (Fig. 2B). To overcome this, a rotation of the imidazole ring around {kappa}1 and {kappa}2 would be required and would permit His47N{delta}1 to interact with the only possible proton donor in the vicinity, which is Asp34O{delta}2. Asp34 could exist in the protonated state because of its configuration around the metal ion binding site, transferring the proton to His47 during catalysis.

Loxosceles spider venom SMases D share high sequence homology, and the amino acids considered to be involved in catalysis are strictly conserved. Structure-based alignment (Fig. 5) and modeling indicate that the five deletions in all other Loxosceles sp. SMases D, except in SMase I from L. laeta venom, result in a shortened surface loop (variable loop or loop E). Additionally, the disulfide bond formed between Cys53 (located in the catalytic loop B) and Cys201 (located in the flexible loop F), present in other Loxosceles sp. SMases D, probably serve as a bridge and bring loops B and F closer together (Figs. 1, 3, and 5).

In conclusion, in bacterial and spider SMases D, interfacial catalysis is mediated by metal ion binding, and two histidine residues are involved in hydrolyzing sphingomyelin and lysophosphatidylcholine via acid base catalysis. Mg2+-dependent SMases probably share a common catalytic mechanism regardless of the species. These results provide structural data important in further dissecting the mechanism of SMases D, in particular, and Mg2+-dependent neutral SMases, in general.


   FOOTNOTES
 
* * 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/). Back

§ Recipients of FAPESP doctoral fellowships. Back

|| Supported by FAPESP and The Welcome Trust. Back

** 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.

1 The abbreviations used are: SMase, sphingomyelinase; SM, sphingomyelin; PLD, phospholipase D. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. I. Polikarpov, A. L. Rojas, and R. A. P. Nagem for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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