Expression, purification, and characterization of a recombinant neutral ceramidase from Mycobacterium tuberculosis.
Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was expressed in Escherichia coli and purified by Ni-Sepharose and gelfiltration. The purified recombinant enzyme showed a single band and a molecular weight estimated to be 71 kDa on SDS-PAGE. It had a pH optimum at 8.0-9.0 and quite broad specificity for various Cers. Of the Cers of different fatty acid moieties tested, those composed of C6-C24 fatty acids were well hydrolyzed, and Cers with mono unsaturated fatty acids were much more hydrolyzed than those with saturated fatty acids. Using N-dodecanoyl-7-nitrobenz-2-oxa-1,3-4-diazole (NBD)-D-erythro-sphingosine (C12-NBD-Cer) as substrates, the reaction followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values for C12-NBD-Cer were 98.7 muM and 21.1 pmol/min respectively. The purified enzyme also catalyzed the synthesis of Cer in vitro, using NBD-labeled dodecanoic acid and Sph as substrates.
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan. nokino@agr.kyushu-u.ac.jp
Nozomu
N
Okino
Rie
R
Ikeda
Makoto
M
Ito
D001393:Azoles / Q000378:metabolism; D002384:Catalysis; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D055576:Neutral Ceramidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009578:Nitrobenzenes / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013110:Sphingosine / Q000378:metabolism; D013379:Substrate Specificity
C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D002518:Ceramides; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D005231:Fatty Acids, Unsaturated; D007850:Lauric Acids; D009578:Nitrobenzenes; D011994:Recombinant Proteins; C030358:lauric acid; D055576:Neutral Ceramidase; D013110:Sphingosine