Purification and characterization of protein PB of betaine reductase and its relationship to the corresponding proteins glycine reductase and sarcosine reductase from Eubacterium acidaminophilum.
Simple complementation assay systems were developed for the substrate-specific proteins PB of glycine reductase, sarcosine reductase, and betaine reductase, in which acetyl phosphate was detected as the product in all three cases. The betaine-specific subunits of protein B (PB betaine) responsible for betaine reductase activity were purified to homogeneity from cells of Eubacterium acidaminophilum. The molecular masses of the two different subunits were 45 kDa and 48 kDa according to SDS/PAGE. The molecular mass of the native protein was about 200 kDa, indicating and alpha 2 beta 2 structure. The glycine-specific protein B (PB glycine) was partially purified and subunits of 47 kDa and 27 kDa were N-terminally sequenced. The latter subunits cross-reacted with antibodies raised against PB betaine and showed high sequence similarity to the 45-kDa and 48-kDa subunits of PB betaine, respectively. [2-14C]Glycine could be covalently coupled to the 47-kDa subunit by treatment with borohydride. By the same procedure, [2-14C]sarcosine labeled a protein of the same size. Like the sarcosine reductase activity, this protein was not present in glycine-grown cells, indicating its specific involvement in sarcosine metabolism. The labile viologen-dependent formate dehydrogenase purified with the respective PB proteins and could be tentatively assigned to a 95-kDa protein.
Meyer
M
M
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Granderath
K
K
Andreesen
J R
JR
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001622:Betaine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005051:Eubacterium / Q000201:enzymology*; D005560:Formate Dehydrogenases / Q000302:isolation & purification; D005998:Glycine / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D012521:Sarcosine / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D001622:Betaine; D005560:Formate Dehydrogenases; D000594:Amino Acid Oxidoreductases; C020815:glycine reductase; C097250:betaine reductase; C097251:sarcosine reductase; D005998:Glycine; D012521:Sarcosine