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 anaerobically grown cultures of the photosynthetic bacterium Rhodopseudomonas viridis to electrophoretic homogeneity 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 lambda EMBL4. 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 previously 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 T7 promoter. Upon induction, transformed F. coli cells accumulate enzymatically active and highly stable R. viridis HSS at levels corresponding to 40-50% of the soluble protein in crude extracts.
Tholl
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Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Germany.
Ober
D
D
Martin
W
W
Kellermann
J
J
Hartmann
T
T
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D003959:Diamines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012241:Rhodopseudomonas / Q000201:enzymology* / Q000235:genetics; D017421:Sequence Analysis; D014166:Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D017931:DNA Primers; D003959:Diamines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C028376:homospermidine synthetase