RGD Reference Report - Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase. - Rat Genome Database

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Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase.

Authors: Brizio, C  Brandsch, R  Bufano, D  Pochini, L  Indiveri, C  Barile, M 
Citation: Brizio C, etal., Protein Expr Purif 2004 Oct;37(2):434-42.
RGD ID: 1547831
Pubmed: PMID:15358367   (View Abstract at PubMed)
DOI: DOI:10.1016/j.pep.2004.06.011   (Journal Full-text)

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%.

Gene Ontology Annotations    Click to see Annotation Detail View

Molecular Function
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
flavin adenine dinucleotide binding  TAS 1547831 RGD 

Molecular Pathway Annotations    Click to see Annotation Detail View

RGD Manual Annotations

Objects Annotated

Genes (Rattus norvegicus)
Dmgdh  (dimethylglycine dehydrogenase)

Genes (Homo sapiens)
DMGDH  (dimethylglycine dehydrogenase)


Additional Information