Purification and Characterization of Homospermidine Synthase in Acinetobacter tartarogenes ATCC 31105

• Published in: Journal of biochemistry (Tokyo) 1993-07, Vol.114 (1), p.45-49
• Main Authors: Yamamoto, Shigeo, Nagata, Satoko, Kusaba, Kaoru
• Format: Article
• Language: English
• Subjects: Acinetobacter - enzymology; Aldehydes - metabolism; Alkyl and Aryl Transferases; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cadaverine - metabolism; Diamines - metabolism; Diamines - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Isoelectric Point; Lathyrus sativus; Ligases; Molecular Sequence Data; Molecular Weight; NAD - metabolism; NAD - pharmacology; NADP - metabolism; Oxidation-Reduction; Putrescine - metabolism; Rhodopseudomonas viridis; Schiff Bases; Substrate Specificity; Sulfhydryl Reagents - pharmacology; Temperature; Transferases - chemistry; Transferases - isolation & purification; Transferases - metabolism
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a124137

Homospermidine synthase, catalyzing the formation of homospermidine [H2N(CH2)4NH-(CH2)4NH2] from putrescine and NAD+ with concomitant liberation of NH3, was purified 600-fold over the crude extract with a yield of about 14% to homogeneity from Acinetobacter tartarogenes ATCC 31105. The enzyme had a native molecular mass of 102 kDa, with a pI of 5.0, and was apparently composed of two identical subunits (52 kDa), suggesting that a single protein catalyzes two serial reactions, oxidation of putrescine to 4-aminobutyral-dehyde and subsequent reduction of the putative Schiff base formed between this aldehyde and a second molecule of putrescine to homospermidine. The Km values for putrescine and NAD+ were 280 and 18 μM, respectively. 1, 3-Diaminopropane and cadaverine were inactive as substrates, and NAD+ could not be replaced by NADP+. 1, 3-Diaminopropane and NADH were potent competitive inhibitors. The enzyme had a pH optimum of 8.7, was most active at 30°C, and required K+ and dithiothreitol for full activity. Putrescine and NAD+ protected the enzyme from the inhibition by thiol reagents. The NH2-terminal amino acid sequence was AQWPVYGKISGPVVI. Some of these properties were compared with those of the homospermidine synthases from a photosynthetic bacterium, Rhodopseudomonas viridis and a plant, Lathyrus sativus.