Purification and characterization of homospermidine synthase in Acinetobacter tartarogenes ATCC 31105.
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-aminobutyraldehyde 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 microM, 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 degrees 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.
Yamamoto
S
S
Faculty of Pharmaceutical Sciences, Okayama University.
Nagata
S
S
Kusaba
K
K
D000150:Acinetobacter / Q000201:enzymology*; D000447:Aldehydes / Q000378:metabolism; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D002103:Cadaverine / Q000378:metabolism; D003959:Diamines / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011700:Putrescine / Q000378:metabolism; D012545:Schiff Bases; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000447:Aldehydes; D003959:Diamines; D012545:Schiff Bases; D013439:Sulfhydryl Reagents; D009243:NAD; C006509:4-aminobutyraldehyde; D009249:NADP; C009475:trimethylenediamine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C028376:homospermidine synthetase; D002103:Cadaverine; D011700:Putrescine