1887

Abstract

PAO1 has two possible catabolic pathways of spermidine and spermine; one includes the and products with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH in PAO1 were characterized and the corresponding gene in this strain identified. The deduced SpdH (620 residues, calculated of 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units (mg protein) and a value of 36 μM. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units (mg protein) and a of 18 μM. Knockout of had no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, when was fused to the polyamine-inducible promoter of , it fully restored the ability of a mutant to utilize spermidine. It is concluded that SpdH can perform a catabolic role , but PAO1 does not produce sufficient amounts of the enzyme to execute this function.

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2006-08-01
2019-11-12
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Resolution of SpdHs purified from PAO1 and BL21(DE3) harbouring plasmid pYI435 ( ), by SDS-PAGE. lane 1, SpdH (0.2 µg) purified from PAO1; lane 2, SpdH (1 µg) purified from E. coli BL21(DE3) harbouring pYI435; lane 3, protein markers: phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa) and catalase (20 kDa). [PDF](46 kb) Consumption of spermidine (A) and spermine (B) by PAO1 and PAO4548 ( ::Gm). Wild-type and mutant cells (1x10 ml ) growing exponentially in MMP containing 20 mM spermidine (inducible substrate) or 20 mM glutamate (non-inducible substrate) as sole carbon and nitrogen sources were transferred to fresh MMP containing 20 mM spermidine (A) or 20 mM spermine (B) and shaken at 37 C. Duplicate portions were removed from cultures at the indicated incubation times and amounts of polyamines were determined by HPCL. Open circles, non-induced PAO1; filled circles, induced PAO1; open squares, non-induced PAO4548; filled squares, induced PAO4548. [PDF](40 kb)

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Resolution of SpdHs purified from PAO1 and BL21(DE3) harbouring plasmid pYI435 ( ), by SDS-PAGE. lane 1, SpdH (0.2 µg) purified from PAO1; lane 2, SpdH (1 µg) purified from E. coli BL21(DE3) harbouring pYI435; lane 3, protein markers: phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa) and catalase (20 kDa). [PDF](46 kb) Consumption of spermidine (A) and spermine (B) by PAO1 and PAO4548 ( ::Gm). Wild-type and mutant cells (1x10 ml ) growing exponentially in MMP containing 20 mM spermidine (inducible substrate) or 20 mM glutamate (non-inducible substrate) as sole carbon and nitrogen sources were transferred to fresh MMP containing 20 mM spermidine (A) or 20 mM spermine (B) and shaken at 37 C. Duplicate portions were removed from cultures at the indicated incubation times and amounts of polyamines were determined by HPCL. Open circles, non-induced PAO1; filled circles, induced PAO1; open squares, non-induced PAO4548; filled squares, induced PAO4548. [PDF](40 kb)

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