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. 2019 Nov 19;9:17016. doi: 10.1038/s41598-019-53171-9

Figure 5.

Figure 5

Site–directed mutagenesis identifies the catalytic residues of BpLOG. The phosphoribohydrolase activity of BpLOG variants with conservative or non-conservative mutations was quantified at 30 °C in the presence of 20 mM of the different nucleotides as substrates. BpLOG variants with conservative variations with AMP (A, B), for 6 (A) or 18 (B) hours, using GMP as substrate with non-conservative (C, D) or conservative (G, H) mutations for 20 min. (C, G) or 40 min. (D, H), BpLOG using CMP as substrate with non-conservative (E, F) or conservative (I, L) for 20 (E, I) or 40 (F, L) min. Mixtures were heat inactivated (AMP, CMP) at 95 °C for 5 min. or inactivated (GMP) with 1 M NaOH. The products were separated by TLC with 1 M NaCl as mobile phase on Cellulose F plastic sheets (AMP, GMP) or with acetone/water 30/70 (v/v) with the addition of 0.2 mol/l NaCl as mobile phase (CMP). The dots were visible under UV light at 264 nm. Arrows indicate the different substrates and the cleaved products. Experiments repeated three times gave identical results.