Aspartate kinase-homoserine dehydrogenase bifunctional enzyme
--Gary J. Muehlbauer, David A. Somers, Benjamin F. Matthews and Burle G. Gengenbach

Aspartate kinase (AK) and homoserine dehydrogenase (HSDH) are enzymes in the aspartate-derived amino acid pathway which leads to the production of lysine, methionine, threonine and isoleucine. Regulatory control of AK and HSDH is mediated through feedback inhibition by end product amino acids. AK catalyzes the first reaction in this pathway and exists in isoforms that are feedback inhibited by lysine, lysine plus S-adenosyl methionine and threonine. Recently in maize, threonine-sensitive AK was shown to copurify with threonine-sensitive HSDH, indicating the possibility of an AK-HSDH bifunctional enzyme (Azevedo et al., Phytochem. 31:373-374, 1992). The putative AK-HSDH bifunctional enzyme was proposed to be a 180 kDa dimer. HSDH catalyzes the first committed step in the synthesis of threonine and exists in threonine-sensitive and insensitive forms. Threonine-sensitive HSDH was previously shown to be a 180 kDa dimer composed of 89 kDa subunits (Walter et al., J. Biol. Chem. 254:1349-1355, 1979). Therefore, it seems likely that threonine-sensitive AK and HSDH exists as a bifunctional enzyme, whereas, lysine-sensitive AK and threonine-insensitive HSDH exist as monofunctional enzymes.

To further investigate this hypothesis we isolated one partial and two full-length cDNAs encoding AK-HSDH from a lgt11 seedling leaf cDNA library using a carrot AK-HSDH cDNA (Weisemann and Matthews, Plant Mol. Biol. 22:  301-312, 1993) as a probe. pAKHSDH1 is a 3178 bp cDNA consisting of a 41 nucleotide 5' untranslated sequence, a 92 amino acid putative transit peptide sequence, an 828 amino acid coding region and a 377 bp 3' untranslated sequence.  pAKHSDH2 is a 3051 bp cDNA consisting of a 49 bp 5' untranslated sequence, a 89 amino acid putative transit peptide sequence, a 828 amino acid coding sequence and a  251 bp 3' untranslated sequence.  The predicted molecular weights for subunits encoded by pAKHSDH1 and pAKHSDH2 were 90,319 and 89,818 Da respectively.  pAKHSDH1 and pAKHSDH2 were 77 and 75% identical at the amino acid level to carrot AK-HSDH, repsectively.  Both clones are divided into three domains; an amino terminal AK domain, a central interface domain, and a carboxy terminal HSDH domain.  Of potential regulatory interest was the conservation of sequences observed in the HSDH domain with four sequence motifs from transmitter modules of prodaryotic two-component regulatory proteins.  Two-component regulatory proteins produce adaptive responses to environmental stimuli via phosphorylation mechanisms.  Therefore, AK-HSDH may possess a phosphorylation mechanism as a way to regulate enzyme activity, sensitivity or both.

To verify that the clones encode functional AK and HSDH activity, a biochemical and immunological study was conducted. Antibodies were raised against a 13-amino acid peptide sequence from pAKHSDH1. AK and HSDH activities were copurified using a Blue Sepharose column. The Blue Sepharose fraction contained only threonine-sensitive AK activity and no lysine-sensitive AK activity, whereas 72% of the HSDH activity in this preparation was threonine sensitive. Threonine-sensitive AK and partially threonine-sensitive HSDH activities migrated to the same position on native PAGE. The pAKHSDH1-derived antibodies cross-reacted on a native protein blot to a protein that corresponded to threonine-sensitive AK and partially threonine-sensitive HSDH activities. The antibodies also cross-reacted with an 89 kDa protein on SDS PAGE, which is the same molecular weight as previously reported for threonine-sensitive HSDH and the same size as the predicted subunit molecular weights for pAKHSDH1 and pAKHSDH2. These data indicated that pAKHSDH1 encodes a subunit of threonine-sensitive AK-HSDH. pAKHSDH2 contains high identity to pAKHSDH1, indicating that it probably also encodes for a subunit of threonine-sensitive AK-HSDH.

RNA blot analyses of AK-HSDH demonstrated hybridization to a single 3.2 kb transcript in embryo, endosperm, leaf and Black Mexican Sweet tissue culture cells. These data demonstrated that threonine-sensitive AK and HSDH activities are encoded by a single transcript. Low stringency hybridizations and washes did not detect smaller transcripts that might encode monofunctional AK or HSDH. These data indicated that AK-HSDH has diverged significantly from monofunctional AK and HSDH.

The chromosomal locations of pAKHSDH1 and pAKHSDH2 were determined using the immortalized F2 population created at the University of Missouri and provided by E. Coe (Gardiner et al., Genetics 134:917-930, 1993). DNA blots containing the immortalized F2 population were hybridized with gene-specific probes from pAKHSDH1 and pAKHSDH2. Using the MAPMAKER computer program (Lander, E et al., Genomics 1:174-181, 1987) and the University of Missouri core RFLP data base, pAKHSDH1 and pAKHSDH2 were positioned on chromosomes 4S and 2L, respectively. pAKHSDH1 was positioned between umc191(gpc1) and umc201(nr) at 7.5 cM and 2.7 cM, respectively. pAKHSDH2 was positioned on chromosome 2L between umc055 and umc139 at 3.6 cM and 4.9 cM, respectively. Nonspecific probes for the partial cDNA clone, pAKHSDH3, detected polymorphisms only on chromosomes 2L and 4S in the same locations as pAKHSDH1 and pAKHSDH2. These blots also contained other monomorphic bands; therefore, an alternate location for pAKHSDH3 may be possible. A gene-specific probe for pAKHSDH3 did not detect a polymorphism between the F2 parents, Tx303 and CO159, with 20 restriction enzymes and it has not been possible to determine the map location of pAKHSDH3.

Further efforts will be directed at isolating the full length cDNA for pAKHSDH3 and determining its map location. However, highest priority will be investigating the potential phosphorylation mechanism of AK-HSDH. 

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