Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803.
A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein)(-1) and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme)(-1) suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.
Engels
A
A
Biologie VIII, Zellphysiologie and Biologie 1, Morphologie der Pflanzen und Feinbau der Zelle, Universität Bielefeld, Germany.
Kahmann
U
U
Ruppel
H G
HG
Pistorius
E K
EK
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000201:enzymology* / Q000648:ultrastructure; D003902:Detergents; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D019281:Dimerization; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis; D017385:Sequence Homology; D012995:Solubility; D013053:Spectrophotometry
D003902:Detergents; D005182:Flavin-Adenine Dinucleotide; D017886:Durapatite; D008058:Dihydrolipoamide Dehydrogenase