Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695.
The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.
Lee
Mon-Juan
MJ
Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.
Huang
Chung-Yu
CY
Sun
Yuh-Ju
YJ
Huang
Haimei
H
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants; D016415:Sequence Alignment; D013095:Spermidine / Q000096:biosynthesis*; D011436:Spermidine Synthase / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D011436:Spermidine Synthase; D013095:Spermidine