Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase.
Dimethylglycine dehydrogenase (Me(2)GlyDH) is a mitochondrial enzyme that catalyzes the oxidative demethylation of dimethylglycine to sarcosine. The enzyme requires flavin adenine dinucleotide (FAD), which is covalently bound to the apoprotein via a histidyl(N3)-(8alpha)FAD linkage. In the present study, the mature form of rat Me(2)GlyDH has been over-expressed in Escherichia coli as an N-terminally 6-His-tagged fusion protein. The over-expressed protein distributed almost equally between the soluble and insoluble (inclusion bodies) cell fraction. By applying the soluble cell lysate to a nickel-chelating column, two fractions were eluted, both containing a nearly homogeneous protein with a molecular mass of 93 kDa, on SDS-PAGE. The first protein fraction was identified by Western blotting analysis as the covalently flavinylated Me(2)GlyDH. It showed optical properties and specific activity (240 nmol/min/mg protein) similar to those of the native holoenzyme. The second fraction was identified as an underflavinylated (apo-) form of Me(2)GlyDH, with a 70% lower specific activity. The recombinant holoenzyme exhibited optimal activity at pH 8.5, an activation energy of about 80 kJ/mol, and two KM values for N,N-dimethylglycine (KM1 = 0.05 mM and KM2 = 9.4 mM), as described for the native holoenzyme. Starting from the inclusion bodies, the unfolded flavinylated enzyme was solubilized by SDS treatment and refolded by an 80-fold dilution step, with a reactivation yield of 50-60%.
Brizio
Carmen
C
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy.
Brandsch
Roderich
R
Bufano
Daniela
D
Pochini
Lorena
L
Indiveri
Cesare
C
Barile
Maria
M
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D050893:Dimethylglycine Dehydrogenase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D024101:Mitochondrial Proteins; D009532:Nickel / Q000737:chemistry; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry
D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C025138:dimethylglycine; D009532:Nickel; D010089:Oxidoreductases, N-Demethylating; D050893:Dimethylglycine Dehydrogenase; C494400:Dmgdh protein, rat; D012521:Sarcosine