Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases

Escherichia coli Cytosolic Glycerophosphodiester Phosphodiesterase (UgpQ) Requires Mg 2+ , Co 2+ , or Mn 2+ for Its Enzyme Activity

“…Starvation for phosphate was provoked by cultivation in a medium containing 0. 16 (1). The phosphate-rich medium contained the same reagents, except for 0.6 mM KH 2 PO 4 .…”

Molecular Cloning, Expression, Function, Structure and Immunoreactivities of a Sphingomyelinase D from Loxosceles adelaida, a Brazilian Brown Spider from Karstic Areas

“…This feature, not present in class I SMases D, diminishes significantly the active-site volume and also alters the inherent flexibility exhibited by the flexible loop. Beyond that, all the structural features observed in class I SMases D are fully conserved and details concerning the action mechanism are well described in Murakami et al, 2005 andMurakami et al, 2006. 3.6 Sphingomyelin-binding mode to SMases D Despite the structure of a SMase D member has been solved, there is neither structural nor biophysical data relating the binding mode of the sphingomyelinase (SM) into the active-site cleft of SMases D. Thus, in order to shed light into the structural determinants for recognition and binding of SM by SMases D, a MD simulation was performed using the crystal structure of the SMase D I and the SM docked into enzyme taking into account the crystallographic position of the sulphate ion, which provides a good notion how the phosphate group from SM is oriented in the active-site cavity.…”

“…SMase D A1 displays a typical TIM ( / ) 8 -barrel fold and its active-site cleft, formed by the metal-binding site (Asp, Glu and Asp) and the two catalytic histidines, is furthermore surrounded by the catalytic loop (residues 46-60), variable loop (residues 167-175), flexible loop (residues 196-203) and other short hydrophobic loops (Figure 8). Based on the current classification of SMases D (Murakami et al, 2006), SMase A1 belongs to class II containing an additional disulphide bridge (Cys53-Cys201), which connects the catalytic loop to flexible loop (Figure 8). This feature, not present in class I SMases D, diminishes significantly the active-site volume and also alters the inherent flexibility exhibited by the flexible loop.…”

“…All other SMases D, such as SMases P1 and P2 from L. intermedia (Tambourgi et al, 1998, Lr1 and Lr2 from L. reclusa and Lb1, Lb2 and Lb3 from L. boneti (RamosCerrillo et al, 2004) belong to class 2, which contains an additional intra-chain disulphide bridge that links the flexible loop with the catalytic loop (Murakami et al, 2006). The class 2 enzymes can be further subdivided into class 2a and class 2b depending on whether they are capable of hydrolysing sphingomyelin or not, respectively (Murakami et al, 2006). One representative of class 2b is the isoform 3 from L. boneti an inactive SMase D isoform (Ramos-Cerrillo et al, 2004) (Table 1).…”

“…But, again, SMase D P1 was more active than A1. Based on sequence and structural similarities, the SMases D can be grouped into two classes depending on the presence of an additional disulphide bridge between the catalytic loop and flexible (Murakami et al, 2006). L. adelaida SMase D A1 is a class II member and conserves all structural features for catalytic activity upon sphingomyelin.…”