Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adeno-syltransferase (MAT), con sists of catalytic alpha(2)/alpha(2) subunits and a noncatalytic beta subun it, believed to have a regulatory function. The full-length cDNA that encod es the beta subunit of human MAT II was cloned and found to encode for a 33 4-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyz e the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, whic h was expressed in Escherichia cell, was recognized by antibodies to the hu man MAT II, to synthetic peptides copying the sequence of native beta subun it protein, and to the r beta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibo dies reacted with protein extracts of E. coli host cells, suggesting that t hese bacteria have no beta subunit protein. Interestingly, the r beta subun it associated with E. coli as well as human MAT a subunits. This associatio n changed the kinetic properties of both enzymes and lowered the K-m of MAT for L-methionine. Together, the data show that we have cloned and expresse d the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity a nd consequently the levels of AdoMet may be modulated in mammalian cells.