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Fig. 3 | BMC Evolutionary Biology

Fig. 3

From: Identification and classification of small molecule kinases: insights into substrate recognition and specificity

Fig. 3

The simple substrate recognition rule revealed through mcBPPS analysis. The residues shown as APH2- or APH3-specific are shown in the respective structure figure panels. a APH2 catalytic site showing the unique residues (carbon atoms colored green) and catalytic aspartates (carbon atoms colored light pink). The receiving hydroxyl group is circled in red. b APH3 catalytic site showing the unique residues (carbon atoms colored blue) and catalytic residues. c Several APH2 and APH3 substrate bound conformations present in PDB are shown schematically. The substrate hydroxyl is circled in red in each case. For each APH2 substrate, a schematic catalytic aspartate (colored light pink) and APH2-speciifc aspartate (colored blue) are shown that provide the substrate binding specificity. For each APH3 substrate, the catalytic aspartate and APH3-conserved arginine are shown schematically as binding to a specific pattern of chemical groups on the substrate. For APH2 substrates, starting from the substrate hydroxyl, the OH-NH2-OH pattern is shown, whereas for APH3, the OH-OH-NH2 pattern is shown. The schematic of PDB structure 3HAV shows that when an APH2 enzyme is presented with APH3 substrate (streptomycin), the substrate still binds in an APH2 recognition pattern (OH-NH2-OH) but without correct stereochemistry of the substrate hydroxyl for substrate phosphorylation to take place. d mcBPPS output showing flanking segments of the DFG motif in a Contrast Hierarchical Alignment (CHA). The CHA shows representative APH2 sequences as the display alignment, all APH2 sequences as foreground alignment (182 sequences) and all ELK sequences as background alignment (15,790 sequences). The foreground and background alignment are shown as residue frequencies below the display alignment. Residue frequencies at each aligned position are given in integer tenths; for example, an ‘8’ indicates that 80–90 % of the sequences in the foreground alignment match the corresponding pattern residue (with ‘!’ indicating 100 %). The first of these ‘residue frequency’ lines reports the virtual number of aligned sequences after down-weighting for redundancy. Directly below this are shown the number of insertions and deletions at each position, again in integer tenths. The black dots above the alignment indicate the pattern positions that were identified by the mcBPPS sampler and which were used to classify the APH2 sequences. To enhance interpretation of the alignment, pattern-matching residues are colored, with biochemically similar residues colored similarly. For example, acidic residues are shown in red, basic residue in cyan and hydrophobic residues in yellow; histidine, glycine and proline are each assigned a unique color. The height of the red bars above the alignment quantify (using a semi-logarithmic scale) the degree to which residue frequencies in the foreground diverge from the corresponding positions in the background at each position. e CHA alignment showing representative bacterial APH3 sequences as display, all bacterial APH3 sequences as foreground (122 sequences) and APH3-ACAD sequences as background (1560 sequences)

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