Purification and characterization of a novel FMN-dependent enzyme. Membrane-bound L-(+)-pantoyl lactone dehydrogenase from Nocardia asteroides.
Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 mumol.min-1.mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN.
Kataoka
M
M
Department of Agricultural Chemistry, Kyoto University, Japan.
Shimizu
S
S
Yamada
H
H
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004790:Enzyme Induction; D004795:Enzyme Stability; D005486:Flavin Mononucleotide / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009616:Nocardia asteroides / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D019946:Propylene Glycol; D011409:Propylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D003902:Detergents; D008565:Membrane Proteins; D011409:Propylene Glycols; D000077423:Polidocanol; D011092:Polyethylene Glycols; D019946:Propylene Glycol; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C073297:pantoyl lactone dehydrogenase; D010100:Oxygen