Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase.
The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to polynucleotide phosphorylase from Escherichia coli. Indeed, like E. coli polynucleotide phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, the E. coli enzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the cloned gpsI gene in E. coli led to an increase in both polynucleotide phosphorylase and guanosine pentaphosphate synthetase activities in the cloning host. The polynucleotide phosphorylase activities of GPSI and of the E. coli enzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and polynucleotide phosphorylase activities.
Jones
G H
GH
Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
Bibb
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C100969:guanosine pentaphosphate synthetase; D011117:Polyribonucleotide Nucleotidyltransferase; D008025:Ligases