Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic α2/α2′ subunits and a noncatalytic β subunit, believed to have a regulatory function. The full-length cDNA that encodes the β subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The β subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native β subunit protein, and to the rβ protein. There is no cross-reactivity between the MAT II α2 or β subunits. None of the anti-β subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no β subunit protein. Interestingly, the rβ subunit associated withE. coli as well as human MAT α subunits. This association changed the kinetic properties of both enzymes and lowered theKm of MAT for l-methionine. Together, the data show that we have cloned and expressed the human MAT II β subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.