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Intramolecular Regulation of the Opposing (p)ppGpp Catalytic Activities of RelSeq, the Rel/Spo Enzyme from Streptococcus equisimilis. Undine Mechold, 2002.Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared . Two major domains of the 739-residue RelSeq protein are defined by limited proteolytic digestion . In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3'-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn2+-dependent 3'-pyrophosphohydrolase . Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein . Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis . Cellular (p)ppGpp accumulation behavior is consistent with these changes . The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro . These assignments can also apply to RelA and SpoT . The ability of RelSeq to mediate (p)ppGpp accumulation during amino acid starvation in S . equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of RelSeq with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E . coli by inhibiting unregulated degradation and restoring regulated synthetic activity . Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins .
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