title: A charge reversal differentiates (p)ppGpp synthesis by monofunctional and bifunctional Rel proteins.
abstract: A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.
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: Divya
authors.initials: D
authors.lastname: Tiwari
authors.forename: Dimple
authors.initials: D
authors.lastname: Rananaware
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; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007477:Ions; D008025:Ligases / Q000737:chemistry* / Q000502:physiology*; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018502:Streptococcus equi / Q000378:metabolism
chemical_list: D017931:DNA Primers; D007477:Ions; D008670:Metals; D004259:DNA-Directed DNA Polymerase; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D008274:Magnesium