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.
Highlights
In the living world, prokaryotes are the most successful due to the way they respond to changes
This proposal was based on the following observations. (p)ppGpp synthesis by the bifunctional Rel proteins from M. tuberculosis (RelMtb) and Streptococcus equisimilis (RelSeq) is inhibited when Mg2ϩ concentration is higher than that of NTPs, which is not the case for proteins employing a dual divalent cation mechanism (16 – 18)
We investiited at Higher Mg2ϩ Concentrations—Rel proteins that are well gated whether Mg2ϩ can confer a similar effect on the synthesis characterized to synthesize and hydrolyze (p)ppGpp contain activity by monofunctional RelAE. coli
Summary
Expression, and Purification—The bifunctional RelMtb consists of 790 amino acids. PppGpp Synthesis Assays—pppGpp synthesis assays were carried out in a 5-l reaction volume containing 0 –50 mM HEPES (pH 8), 100 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2, 1 mM GTP, 1 mM ATP, and 1 Ci of [␥-32P]ATP and 5 M Rel proteins at 37 °C for 30 min. We investiited at Higher Mg2ϩ Concentrations—Rel proteins that are well gated whether Mg2ϩ can confer a similar effect on the synthesis characterized to synthesize and hydrolyze (p)ppGpp contain activity by monofunctional RelAE. The sequence alignment displayed several conserved motifs across species, our attempt to search for motifs that distinguish the bifunctional and monofunctional proteins led to the identification of a unique charge reversal in a conserved motif in the (p)ppGpp synthesizing domains. (Lys-350) residues in the RXKD motif of bifunctional RelMtb were mutated to Glu and Asp, respectively.
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