Mutation of N406 in the NPPY motif abolishes MTase and cleavage activity.

<p>(A) Fluorescence spectra of WT and N406A RM.BpuSI. The high similarity of the two spectra indicated that mutation N406A does not induce significant conformation change to RM.BpuSI. Titration of SAM or SIN into mutant N406A does not show specific changes in fluorescence (data not shown). (B) Titration of SAM or SIN into WT RM.BpuSI in the presence of 400 μM ANS. The data points are fitted to a hyperbolic function and K_d values are found to be 76.4 and 71.1 μM for SAM and SIN, respectively. (C) Two-fold dilutions of WT or N406A RM.BpuSI was incubated with 1 μg of λ DNA in the presence or absence of 160 μM SAM at 37°C for 1 h. While WT exhibits enhanced cleavage activity in the presence of SAM, mutant N406A does not exhibit cleavage activity in the presence or absence of SAM.