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Identification of new sphingomyelinases D in pathogenic fungi and other pathogenic organisms.

Dias-Lopes C, Neshich IA, Neshich G, Ortega JM, Granier C, Chávez-Olortegui C, Molina F, Felicori L - PLoS ONE (2013)

Bottom Line: We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site.Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus.Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

ABSTRACT
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

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Related in: MedlinePlus

SMD-tail contacts with inner β-strands and loop contact densities in the L. laeta SMase D.(A) Scheme for SMase D secondary structure elements, as described in Murakami et al. (2005) and indicating the SMD-tail (orange), the catalytic, flexible and variable loops (shown in blue, green and purple, respectively), the inner β-strands (yellow) and α-helices (light pink) numbered from 1 to 8. The position of the interaction partners of the SMD-tail (shown in red) and the loops with more than 3 residues coded from A to K. (B) Contact density values obtained by STING contacts for each loop and the entire SMD-tail, the SMD-tail key residues (271-274, 277, 279 and 280) and the ratio for all residues in the protein. (C) Comparison of contact energy density values for the SMD-tail to the entire protein and each type of contact.
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pone-0079240-g003: SMD-tail contacts with inner β-strands and loop contact densities in the L. laeta SMase D.(A) Scheme for SMase D secondary structure elements, as described in Murakami et al. (2005) and indicating the SMD-tail (orange), the catalytic, flexible and variable loops (shown in blue, green and purple, respectively), the inner β-strands (yellow) and α-helices (light pink) numbered from 1 to 8. The position of the interaction partners of the SMD-tail (shown in red) and the loops with more than 3 residues coded from A to K. (B) Contact density values obtained by STING contacts for each loop and the entire SMD-tail, the SMD-tail key residues (271-274, 277, 279 and 280) and the ratio for all residues in the protein. (C) Comparison of contact energy density values for the SMD-tail to the entire protein and each type of contact.

Mentions: It has previously been reported that, along with the fairly well conserved active site, SMases D have a conserved C-terminal motif (without, as of now, any known function) that distinguishes them from the GDPD enzyme family. This SMD-tail, which is located after the last α-helix of the TIM barrel in the C-terminus of SMase D, contains the ATXXDNPW motif, which is well conserved in most of the sequences (Figure 2). Due to its strong conservation, the SMD-tail could be expected to play an important functional or structural role. By analyzing the L. laeta SMase D (PDB 1XX1, chain A) structure using the STING Java Protein Dossier, we found several pieces of evidence indicating the involvement of the SMD-tail in SMase D structure stabilization. Namely, these residues establish a significant number of contacts directly with residues located at seven (out of eight) inner β-strands from the TIM barrel structure, all being on the side of the protein opposite to the catalytic site. The two residues that established the energetically strongest contacts were Asp277 and Trp280. Trp280, as predicted by STING (Figure S1), establishes a hydrogen bond network with Glu159, Asn225 and Asn278 that is mediated by two water molecules. There was a nearly parallel aromatic stacking with His191 and another stacking with residue Tyr128. Hydrophobic contacts were predicted involving Trp193, Gly162, Val161, Lys160, Val130 and Tyr128. Asp277, another highly conserved residue in the SMD-tail motif, also establishes important contacts, predominantly ionic interactions with Arg271, a hydrogen bond (intermediated by two water molecules) with Trp8 from the N-terminal β-sheet hydrogen bonds with Pro279 and Thr274 and hydrophobic contacts with Pro6 (Figure S1). Thus, the conservation of the SMD-tail throughout the family is possibly due to its significant importance in the structural stabilization of inner barrel β-sheets. To strengthen this hypothesis, the contact density and energies established among the enzyme residues were calculated. Both the density of the contacts and the contact energy density are higher in the SMD-tail compared to the entire protein (e.g., 10.7 versus 3.57, as shown in Figure 3B). When compared to all SMase D loops, the SMD-tail is still better with regards to the contact density (Figure 3B). However, the B loop, which contains the catalytic His12, also had a high contact ratio. Considering the distinct types of contacts, there was an average of 2.4 hydrogen bonds per residue in the SMD-tail, whereas this value is much lower when the entire protein was considered (1.5), as shown in Figure 3C. A similar trend was observed for charged attractive and hydrophobic interactions.


Identification of new sphingomyelinases D in pathogenic fungi and other pathogenic organisms.

Dias-Lopes C, Neshich IA, Neshich G, Ortega JM, Granier C, Chávez-Olortegui C, Molina F, Felicori L - PLoS ONE (2013)

SMD-tail contacts with inner β-strands and loop contact densities in the L. laeta SMase D.(A) Scheme for SMase D secondary structure elements, as described in Murakami et al. (2005) and indicating the SMD-tail (orange), the catalytic, flexible and variable loops (shown in blue, green and purple, respectively), the inner β-strands (yellow) and α-helices (light pink) numbered from 1 to 8. The position of the interaction partners of the SMD-tail (shown in red) and the loops with more than 3 residues coded from A to K. (B) Contact density values obtained by STING contacts for each loop and the entire SMD-tail, the SMD-tail key residues (271-274, 277, 279 and 280) and the ratio for all residues in the protein. (C) Comparison of contact energy density values for the SMD-tail to the entire protein and each type of contact.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815110&req=5

pone-0079240-g003: SMD-tail contacts with inner β-strands and loop contact densities in the L. laeta SMase D.(A) Scheme for SMase D secondary structure elements, as described in Murakami et al. (2005) and indicating the SMD-tail (orange), the catalytic, flexible and variable loops (shown in blue, green and purple, respectively), the inner β-strands (yellow) and α-helices (light pink) numbered from 1 to 8. The position of the interaction partners of the SMD-tail (shown in red) and the loops with more than 3 residues coded from A to K. (B) Contact density values obtained by STING contacts for each loop and the entire SMD-tail, the SMD-tail key residues (271-274, 277, 279 and 280) and the ratio for all residues in the protein. (C) Comparison of contact energy density values for the SMD-tail to the entire protein and each type of contact.
Mentions: It has previously been reported that, along with the fairly well conserved active site, SMases D have a conserved C-terminal motif (without, as of now, any known function) that distinguishes them from the GDPD enzyme family. This SMD-tail, which is located after the last α-helix of the TIM barrel in the C-terminus of SMase D, contains the ATXXDNPW motif, which is well conserved in most of the sequences (Figure 2). Due to its strong conservation, the SMD-tail could be expected to play an important functional or structural role. By analyzing the L. laeta SMase D (PDB 1XX1, chain A) structure using the STING Java Protein Dossier, we found several pieces of evidence indicating the involvement of the SMD-tail in SMase D structure stabilization. Namely, these residues establish a significant number of contacts directly with residues located at seven (out of eight) inner β-strands from the TIM barrel structure, all being on the side of the protein opposite to the catalytic site. The two residues that established the energetically strongest contacts were Asp277 and Trp280. Trp280, as predicted by STING (Figure S1), establishes a hydrogen bond network with Glu159, Asn225 and Asn278 that is mediated by two water molecules. There was a nearly parallel aromatic stacking with His191 and another stacking with residue Tyr128. Hydrophobic contacts were predicted involving Trp193, Gly162, Val161, Lys160, Val130 and Tyr128. Asp277, another highly conserved residue in the SMD-tail motif, also establishes important contacts, predominantly ionic interactions with Arg271, a hydrogen bond (intermediated by two water molecules) with Trp8 from the N-terminal β-sheet hydrogen bonds with Pro279 and Thr274 and hydrophobic contacts with Pro6 (Figure S1). Thus, the conservation of the SMD-tail throughout the family is possibly due to its significant importance in the structural stabilization of inner barrel β-sheets. To strengthen this hypothesis, the contact density and energies established among the enzyme residues were calculated. Both the density of the contacts and the contact energy density are higher in the SMD-tail compared to the entire protein (e.g., 10.7 versus 3.57, as shown in Figure 3B). When compared to all SMase D loops, the SMD-tail is still better with regards to the contact density (Figure 3B). However, the B loop, which contains the catalytic His12, also had a high contact ratio. Considering the distinct types of contacts, there was an average of 2.4 hydrogen bonds per residue in the SMD-tail, whereas this value is much lower when the entire protein was considered (1.5), as shown in Figure 3C. A similar trend was observed for charged attractive and hydrophobic interactions.

Bottom Line: We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site.Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus.Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

ABSTRACT
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

Show MeSH
Related in: MedlinePlus