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

Abstract

Simple complementation assay systems were developed for the substrate-specific proteins P, 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, responsible for betaine reductase activity were purified to homogeneity from cells of Eubucterium ueidam~noph~lum. 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 u2P2 structure. The glycine-specific protein B (P, gl,c,,J 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, and showed high sequence similarity to the 45-kDa and 48-kDa subunits of P, hcf.,inc, respectively. 12-"C]Glycine could be covalently coupled to the 47-kDa subunit by treatment with borohydride. By the same procedure, [2-"C]sarcosine labeled a protein of the same size. Like the sarcosine reductase activity, this protein was not present in glycinegrown cells, indicating its specific involvement in sarcosine metabolism. The labile viologen-dependent formate dehydrogenase purified with the respective P,, proteins and could be tentatively assigned to a 95-kDa protein.Keywords: Eubacterium trcidaminophiluin ; glycine reductase ; sarcosine reductase ; betaine reductase ; formate dehydrogenase.Glycine can be utilized by Eubucterium acidaminophilum as sole carbon source by the action of glycine decarboxylase and glycine reductase 11 1. The oxidation of glycine to S,lO-methylenetetrahydrofolate, ammonia, and reduced pyridine nucleotides is performed by glycine decarboxylase. The dihydrolipoamide dehydrogenase (P,) usually involved in the total reaction is not part of the latter enzyme complex in E. acidurninophilum 12. 31. Its function is taken over by a thioredoxin system, catalyzing the same, but NADP(H)-specific reaction that channels the electrons directly to the glycine reductase proteins 13, 41. The glycine reductase of E. acidurnirzophilum consists of at least three proteins (Scheme 1) : the selenocysteine-containing protein A (PA) ; the substrate-binding protein B (P,) ; the acetyl phosphate-forming protein C (P,) ( 5 -7 1. The 5,1O-methylenetetrahydrofolate formed by glycine oxidation is further oxidized in two steps by methylenetetrahydrofolate dehydrogenase and formate dehydrogenase to CO:. Both enzymes are specific for NADP(H) in E. reductase is also present in cells grown on sarcosine or betaine 181 in contrast to the situation in betaine-grown cells of Closrridium litorule, as revealed in this study, where only a betaine reductase, but no glycine reductase activity could be determined. Antibodies directed against P,, and P,. of E. uciduminophilum grown on glycine cross-react with homologous proteins of E. ncidaminophilurn and C. litorale grown on betaine [S, 61, indi-cating that betaine reduction occurs by an enzyme system analogous to glycine ...

Tissierella creatinophila sp. nov., a Gram-positive, anaerobic, non-spore-forming, creatinine-fermenting organism

“…The glycine reductase of all species studied, such as Clos tr idium s t icklandii, Clos tr idium spor ogenes, E. acidaminophilum, Clostridium litorale and Clostridium histolyticum involve selenocysteine-containing proteins, explaining the selenium-dependence of growth (Andreesen, 1994 ;Kreimer & Andreesen, 1995). The reduction of betaine, sarcosine and glycine in E. acidaminophilum involves only substrate-specific proteins that differ in sensitivity towards hydroxylamine (Andreesen, 1994;Meyer et al, 1995). In comparison to E. acidaminophilum, the reductase activities in strain KRE 4T were quite low in extracts.…”

“…These two different metabolic pathways should be distinguishable by the influence of hydroxylamine on the activity of creatineand sarcosine reductase. In contrast to sarcosine and glycine, creatine cannot form a Schiff base with a carbonyl group of the substrate-specific protein to activate the C-N bond, as also observed for betaine (Meyer et al, 1995). Hydroxylamine would form an oxime with the potential carbonyl group, thereby rendering the respective reductase sensitive.…”

“…Creatine is an important natural substance (Shimizu et al, 1986) which might allow the evolution of a specific reductase of its own, as is found for glycine, sarcosine and betaine (Hormann & Andreesen, 1989;Meyer et al, 1995) but not for dimethylglycine (Andreesen, 1994). For this reason, highly specific conditions were chosen for enrichment with creatinine and formate as substrates.…”

“…In addition, a non-radioactive enzyme assay was used by measuring the formation of acetyl phosphate (Meyer et al, 1995).…”

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

“…Anaerobically, catabolic pathways containing GB reductase cleave GB to acetate and trimethylamine (TMA) as excreted products. GB reduction is often considered the sole fate of the compound under anaerobic conditions (17)(18)(19). However, there have been sporadic reports in the literature that GB reduction is not the only route of microbial GB catabolism under anoxic conditions.…”