c Cadherin Fla TOPRO-3 Merge S227A H229A H272A D276A s217A membrane(Fig. 2B). This result confirmed a previous report (17. approximately 30% of a wild type specific activity, suggesting that Moreover, all mutants have a normal cellular distribution, compared although both serines are located next to HHd motifs(Fig 1A), they with WT( Fig 2B and C), indicating the normal enzyme topology is still have a different impact on catalytic domain formation. It has been maintained in the"dead"enzyme suggested that S181 in LPPl, analogous to S283 in SMSI and S227 in We also prepared Flag-tagged wild type SMS2 as well as mutants SMSZ, can form a hydrogen bond with the phosphate group while it analogous to those described above for SMS1( Fig. 3). Similarly, is in the active site instead of directly participating in catalysis [ 19]. individual mutations of the SMSz HHD triad all abolished SMS2 Therefore, it is not surprising that $227A is not completely activity as opposed to wild type SMS2 and the control(S217A) necessary for SMs activity. there is likely a neighboring residue nutant(Fig 3A). Interestingly, one difference between SMSI and within the tertiary structure of SMS2 which is functionally equi SMS2 is that SMS1-S283A has no activity, while SMS2-S227A retains valent to $227.membrane (Fig. 2B). This result confirmed a previous report [17]. Moreover, all mutants have a normal cellular distribution, compared with WT (Fig. 2B and C), indicating the normal enzyme topology is still maintained in the “dead” enzyme. We also prepared Flag-tagged wild type SMS2 as well as mutants analogous to those described above for SMS1 (Fig. 3). Similarly, individual mutations of the SMS2 HHD triad all abolished SMS2 activity as opposed to wild type SMS2 and the control (S217A) mutant (Fig. 3A). Interestingly, one difference between SMS1 and SMS2 is that SMS1-S283A has no activity, while SMS2-S227A retains approximately 30% of a wild type specific activity, suggesting that although both serines are located next to HHD motifs (Fig. 1A), they have a different impact on catalytic domain formation. It has been suggested that S181 in LPP1, analogous to S283 in SMS1 and S227 in SMS2, can form a hydrogen bond with the phosphate group while it is in the active site instead of directly participating in catalysis [19]. Therefore, it is not surprising that S227A is not completely necessary for SMS activity. There is likely a neighboring residue within the tertiary structure of SMS2 which is functionally equi￾valent to S227. Fig. 3 (continued). 614 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617