Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthetic activities on Escherichia coli SpoT domains.
Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5'-diphosphate 3'-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, alpha-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further alpha-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA -, spoT- double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).
Fujita
Chizuko
C
Department of Chemistry, Faculty of Science, Nara Women's University, Japan.
Nishimura
Akiko
A
Iwamoto
Ryoko
R
Ikehara
Kenji
K
D015153:Blotting, Western; D003470:Culture Media; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006159:Guanosine Tetraphosphate / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry
D003470:Culture Media; D017931:DNA Primers; D006159:Guanosine Tetraphosphate; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D012697:Serine Endopeptidases; D011755:Pyrophosphatases