A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

Abstract

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

Structural Requirements for Catalysis and Membrane Targeting of Mammalian Enzymes with Neutral Sphingomyelinase and Lysophospholipid Phospholipase C Activities

“…Based on this prediction and supporting experimental evidence (4,22,23) the function of several residues in the B. cereus enzyme have been suggested. is not carried out by a His in a similar position in the mammalian sequence but by a residue unique to eukaryotic enzymes (7).…”

“…Subsequent site-directed mutagenesis ( Table II) has shown that mutations His136Ala and His272Ala at positions corresponding to general base and acid in other phosphodiesterases, resulted in a great reduction of the enzyme activity consistent with their proposed function. Mutational analysis of the corresponding residues in bacterial NSM and DNAse I had a similar impact on activity of these enzymes (4). It has also been reported (10) that the His272Asn mutation in the human enzyme resulted in a loss of NSM activity, as determined in transiently transfected HEK 293 cells.…”

“…Based on this comparison, it is also likely that all enzymes share the same catalytic mechanism i.e. general acid/base mechanism involving two histidine residues (4). Chemical modification of histidine residues and the substrate protection experiments demonstrated that the murine enzyme contains essential histidines which are present within the active site ( Fig.…”

“…Furthermore, the 3-D fold of the B. cereus enzyme has been predicted and modeled using the structure determined for DNAse I (4). Based on this prediction and supporting experimental evidence (4,22,23) the function of several residues in the B. cereus enzyme have been suggested.…”

The Inositol Polyphosphate 5-Phosphatases and the Apurinic/Apyrimidinic Base Excision Repair Endonucleases Share a Common Mechanism for Catalysis

“…1). It had previously been noted that the AP-endonucleases share sequence similarity with the endonuclease domain of long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons and sphingomyelinases (36,37). A more recent sequence analysis identified the conservation of fold and catalytic residues between the Mg 2ϩ -dependent endonucleases and a variety of signaling proteins including the inositol polyphosphate 5-phosphatases and the yeast carbon catabolite repressor protein Ccr4p (38).…”