title: The significance of EXDD and RXKD motif conservation in Rel proteins.
abstract: Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3'-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg(2+) due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.
authors.affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Mathew
authors.initials: M
authors.lastname: Sajish
authors.forename: Sissy
authors.initials: S
authors.lastname: Kalayil
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Verma
authors.forename: Vinay Kumar
authors.initials: VK
authors.lastname: Nandicoori
authors.forename: Balaji
authors.initials: B
authors.lastname: Prakash
mesh_terms: D020816:Amino Acid Motifs; D055162:Biocatalysis; D017124:Conserved Sequence; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D009711:Nucleotides; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases