Purification, molecular cloning and expression in Escherichia coli of homospermidine synthase from Rhodopseudomonas viridis

Homospermidine synthase (HSS) catalyzes the synthesis of the polyamine homospermidine from 2 mol putrescine in an NAD+-dependent reaction. In this study, the enzyme was purified from anaerobi-cally grown cultures of the photosynthetic bacterium Rhodopseudomonus viridis to electrophoretic homo-geneity using a three-step procedure. The enzyme was shown to be a homodimer of 52-kDa subunits. Six endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers designed against these peptides, specific PCR products from R. viridis DNA were obtained that were used as hybridization probes to isolate the hss gene from a library constructed in AEMBL4. The hss gene and flanking regions were sequenced and were shown to exist as a single copy in the R. viridis genome. HSS is translated from a monocistronic mRNA and possesses no detectable similarity to pre-viously sequenced gene products. Escherichia coli, which lacks HSS activity, was transformed with an expression plasmid containing the hss coding region under the control of a bacteriophage T, promoter. Upon induction, transformed E. coli cells accumulate enzymatically active and highly stable R. viridis HSS at levels corresponding to 40-50 % of the soluble protein in crude extracts