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

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta 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 beta 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 beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association 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 expressed the human MAT II beta 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.     

Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic alpha(2) and regulatory beta subunits. The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT vector harboring full-length cDNA that encodes for the MAT II alpha(2) or beta subunits. Expression of the His-tagged recombinant alpha(2) (ralpha(2)) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in the K(m) for L-methionine (L-Met) from 15 microM (endogenous MAT II) to 75 microM (ralpha(2)), and with the apparent existence of two kinetic forms of MAT in the transfected COS-1 cell extracts. By contrast, expression of the recombinant beta (rbeta) subunit had no effect on the K(m) for L-Met of the endogenous MAT II, while it did cause an increase in both the V(max) and the specific activity of endogenous MAT. Co-expression of both ralpha(2) and rbeta subunits resulted in a significant increase of MAT specific activity with the appearance of a single kinetic form of MAT (K(m) = 20 microM). The recombinant MAT II alpha(2) and rbeta subunit associated spontaneously either in cell-free system or in COS-1 cells co-expressing both subunits. Analysis of nickel-agarose-purified His-tagged ralpha(2) subunit from COS-1 cell extracts showed that the beta subunit co-purified with the alpha(2) subunit. Furthermore, the alpha(2) and beta subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for alpha(2) and beta MAT subunit association. In addition, the beta subunit regulated MAT II activity by reducing its K(m) for L-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II beta subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.     

Biochemical basis for the dominant inheritance of hypermethioninemia associated with the R264H mutation of the MAT1A gene. A monomeric methionine adenosyltransferase with tripolyphosphatase activity

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.