Fig. 2

Structural motifs of GAR and related proteins. a Alignment of GAR with related proteins. The two amino acids that differ between GAR variants (L9I, T75A) are framed in black. The ORF from Luteimonas sp. 83-4 (CP029556) with 100% identity to GAR has no annotation. OrfPa105 (AJ786649) from P. aeruginosa 105MG and the ORF from P. aeruginosa S742_C15_BS without annotation (NFFO01000062.1) are identical. They differ by two nucleotides and one amino acid (T75A) from GAR. The two ORFs from S. enterica ADRDL-2811 (AAKHBQ010000151.1) and S. enterica subsp. enterica serovar Johannesburg FSIS1609335 (AAIUOI010000042.1) are identical to each other, annotated as hypothetical proteins and differ by two nucleotides and one amino acid (L9I) from GAR. Tunicamycin resistance protein TmrB as harboured by Bacillus subtilis (WP_003246258.1), the C-terminal membrane anchor is underlined [19], TmrD from Deinococcus radiodurans (WP_010888058.1) [20]. The N-terminal Walker A motif occurs in TmrB and in GAR, but the absence of the membrane anchor hints to a cytoplasmic location of GAR. The NTP-binding motif G/AXXGXGKT/S (Walker A or P-loop) is marked by red rectangles [18, 21, 22]. Searching NCBI’s conserved domain database revealed an AAA domain (ATPases Associated with diverse cellular Activities, pfam13238) within GAR and similarity to gluconate kinases (COG3265). GAR harbours no Walker B motif, but two potential DxD motifs, which could be responsible for Mg²⁺ binding [23], marked by blue rectangles. b Protein structure model of GAR. According to the structure model, the first DxD motif (aa 31–33) is located in the same cavity as the P-loop and thus more likely to participate in the NTP-binding and hydrolysis. GAR is shorter than other aminoglycoside phosphotransferases (APHs) and seems to contain five N-terminal parallel β-sheets, while APHs contain five anti-parallel β-sheets in their N-terminal domain [24, 25]