Abstract
Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.
Highlights
Sphingomyelinases (SMases) are enzymes that catalyze the hydrolysis of sphingomyelin (SM) to generate ceramide (Cer) and phosphocholine [1,2,3,4]
We find that the RsASML protein, unlike Acid SMase (aSMase), cannot hydrolyze SM to Cer but appears to be an ATP diphosphohydrolase, which catalyzes the hydrolysis of ATP and ADP to AMP
To aid in structurally defining the aSMase protein fold, we searched for bacterial aSMase-like proteins that shared sequence homology with human aSMase and could be used for structural studies
Summary
Sphingomyelinases (SMases) are enzymes that catalyze the hydrolysis of sphingomyelin (SM) to generate ceramide (Cer) and phosphocholine [1,2,3,4]. Bacterial homologues of neutral SMases have been identified in many pathogens including Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Listeria ivanovii and Streptomyces griseocarneus [1,3,4,24,25]. In these pathogenic bacteria, the secreted neutral SMases are toxins that catalyze the hydrolysis of SM on the outer plasma membrane leaflet of erythrocytes and lymphocytes, causing hemolysis, lymphotoxicity, and septicemia [24,25,26]. We identify and biochemically characterize the gene RSp1609, referred to as RsASML, as the first bacterial aSMase-like protein from the deadly plant pathogen R. solanacearum. The identification of a bacterial aSMase-like protein suitable for X-ray crystallography studies may aid in future studies defining the structural features of the aSMase protein superfamily
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