PURIFICATION AND CHARACTERIZATION OF PROTEIN P-B 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 P-B 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 (P-B 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 an alpha(2) beta(2) structure. The glycine-specific protein B (P-B 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 P-B betaine and showed high sequence similarity to the 45-kDa and 48-kDa subunits of P-B betaine, respectively. [2-C-14]Glycine could be covalently coupled to the 47-kDa subunit by treatment with borohydride. By the same procedure, [2-C-14]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 P-B proteins and could be tentatively assigned to a 95-kDa protein.