Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter...

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• The HP0832 (speE) gene in Helicobacter pylori strain 26695 encodes a putative spermidine synthase.
• Spermidine synthase is a key enzyme in the polyamine biosynthetic pathway, catalyzing spermidine production from putrescine and decarboxylated S-adenosylmethionine (dcSAM).
• The HP0832 protein shows low sequence identity to spermidine synthases from other organisms.

Purpose of the Study:

• To clone, express, and purify the HP0832 gene product from H. pylori.
• To confirm the enzymatic activity and substrate specificity of the recombinant HP0832 protein.
• To investigate the role of spermidine synthase in H. pylori polyamine metabolism.

Main Methods:

• Cloning of the HP0832 open reading frame into the pQE30 vector.
• Overexpression in Escherichia coli and purification of the N-terminally 6xHis-tagged protein using Ni-NTA affinity chromatography.
• Enzymatic assays with putrescine and dcSAM to confirm spermidine synthase activity.
• Detection of endogenous H. pylori spermidine synthase using specific antiserum.

Main Results:

• The recombinant HP0832 protein (31.9 kDa) was successfully purified with a yield of 15 mg/L.
• Enzymatic assays confirmed the spermidine synthase activity of the purified protein.
• Substrate specificity studies indicated that spermidine cannot replace putrescine as an acceptor.
• H. pylori strain 26695 contains putrescine and spermidine in a 1:3 molar ratio, with no detectable spermine or norspermidine.

Conclusions:

• The HP0832 gene product is a functional spermidine synthase in H. pylori.
• The spermidine biosynthetic pathway likely accounts for the primary polyamines in H. pylori strain 26695.
• The findings provide insights into polyamine metabolism in this important human pathogen.