Proteome of proline betaine-grown Eubacterium limosum ATCC 8486

Proline betaine, and to a much lesser extent, N-methyl L-proline, are found in citrus fruits and often present in the human metabolome. However, most of the N-methyl L-proline found in an animal model metabolome is produced by the microbiota, yet organisms and enzymes capable of producing N-methyl proline in the anoxic intestine have been unknown. The anaerobe Eubacterium limosum ATCC 8486 makes acetate and butyrate from various substrates and is found in the human intestine. We found that this organism demethylates proline betaine and excretes N-methyl proline during growth. The proteome of proline betaine-grown Eubacterium limosum was obtained in order to identify enzymes required for growth on proline, in particular to identify components that are unique to growth on proline in comparison to other substrates for acetogenesis, such as lactic acid. Comparison of the proteomes of the bacteria on proline betaine and lactic acid led to identification of the proteins involved in a proline betaine:tetrahydrofolate methyltransferase system which was biochemically verified. The key proline betaine demethylating enzyme is a member of the widespread TMA methyltransferase protein superfamily.