High-level expression and purification of rat monoamine oxidase A (MAO A) in Pichia pastoris: comparison with human MAO A.
The high-level heterologous expression in Pichia pastoris, purification and characterization of recombinant membrane-bound rat liver monoamine oxidase A (MAO A) are described. A 1-L culture of cells produces approximately 700 U of rat MAO A activity. The rat MAO A activity is found in outer mitochondrial membrane of the cell. Using a modification of the human MAO A purification procedure, approximately 200mg of recombinant rat MAO A is purified in a 43% yield and exhibits a molecular weight of approximately 60,000 kDa on SDS-PAGE. The purified enzyme contains a covalently bound FAD and forms a N(5) flavocyanine adduct on inhibition by clorgyline. Edman sequencing shows that the amino terminus of rat MAO A is blocked at an N-terminal threonyl residue. The purified rat enzyme exhibits a higher thermal stability than does purified human MAO A. Compared with human MAO A, rat MAO A oxidizes serotonin or kynuramine with twofold higher k(cat)/K(m) values, oxidizes phenethylamine with a 6.7-fold higher catalytic efficiency and benzylamine with a approximately 40-fold higher catalytic efficiency. Although approximately 90% identical in sequence to human MAO A, rat MAO A is a more efficient catalyst for amine neurotransmitter oxidation.
Wang
Jin
J
Department of Biochemistry, Emory University, Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322, USA.
Edmondson
Dale E
DE
D000818:Animals; D001596:Benzylamines / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008995:Monoamine Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010627:Phenethylamines / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D001596:Benzylamines; D010627:Phenethylamines; C029261:phenethylamine; C030796:benzylamine; D008995:Monoamine Oxidase