--S.B. Dotson, D.A. Somers and B.G. Gengenbach
Aspartate kinase is the first enzyme in the aspartate-derived amino acid biosynthesis pathway which leads to the production of lysine, methionine, threonine and isoleucine. Two lysine plus threonine (LT) resistant mutants previously selected from tissue culture overproduce free threonine compared to wildtype, implicating an alteration in the regulation of aspartate-derived amino acid synthesis (Hibberd et al., Proc. Natl. Acad. Sci. 79:559-563, 1982; T.J. Diedrick, Ph.D. Thesis, U of M, 1984). These mutants represent dominant alleles of two different genes designated Ltr*-1 and Ltr*-2 (Frisch and Gengenbach, MNL 60:115, 1986). The objectives of this research were to purify and characterize wildtype aspartate kinase to determine its regulatory characteristics and to determine whether the Ltr*-1 and Ltr*-2 mutations encode lysine-insensitive forms of AK that allow threonine overproduction.
Aspartate kinase was purified to greater than 1200-fold from wildtype 'Black Mexican Sweet' corn suspension culture cells (Table 1). Holoenzyme molecular weight of corn aspartate kinase determined by gel filtration was 255,000Mr. Separation by anion exchange resolved corn aspartate kinase activity into two isoforms. Both isoforms were similarly inhibited by lysine and lysine analogs. Threonine had no effect on corn aspartate kinase activity either alone or in combination with lysine. Aspartate kinase activity staining of native PAGE gels indicated that corn aspartate kinase disaggregated to ca. 120,000Mr species from the 255,000 Mr species originally detected by gel filtration. The aspartate-dependent aspartate kinase activity visualized on native gels was excised and rerun on SDS PAGE gels. Three bands of ca. 49-63 kDa were resolved from this highly purified preparation.
Table 1. Purification of aspartate kinase from Black Mexican Sweet Corn cultures.
Characterization of LT resistant corn mutants has been somewhat limited by difficulties in deriving homozygous kernels that germinate and develop normally. This summer we isolated unpollinated immature ears from homozygous Ltr*-2 plants and heterozygous Ltr*-1 plants in A619 background. Aspartate kinase was purified more than 1000-fold from each mutant and from wildtype A619 using the procedure developed for corn cell suspension culture aspartate kinase. Only one peak of aspartate kinase activity was resolved by ion exchange chromatography for Ltr*-2 and A619 (Fig. 1). Furthermore, the shapes of the ion exchange peak of aspartate kinase from A619 and Ltr*-2 were similar. Lysine inhibition of aspartate kinase activity from homozygous Ltr*-2 was substantially different compared with A619 aspartate kinase. The activity in Ltr*-2 required approximately 760µM lysine for 50% inhibition compared to approximately 10µM lysine for the wildtype, indicating about a 76-fold reduction in sensitivity to feedback regulation by lysine.
Figure 1. Anion exchange (FPLC) elution profiel of aspartate kinase activity of A619 (•), Ltr*-1 (o), and Ltr*-2 (x).
Analysis of aspartate kinase from heterozygous Ltr*-1 plants was more complicated because of the presence of the wildtype and mutant alleles in this material. Ltr*-1 aspartate kinase showed broader elution profile on ion exchange with the majority of activity eluting later than Ltr*-2 and A619 AK. The Ltr*-1 aspartate kinase preparation also exhibited reduced lysine feedback inhibition. Early and late eluting Ltr*-1 aspartate kinase activity was inhibited 50% by 38µM and 25µM lysine, respectively, in contrast to 10µM lysine required for 50% inhibition of aspartate kinase from wildtype A619.
The aspartate kinase isoforms encoded by Ltr*-1 and Ltr*-2
were apparently copurified by our procedures. We assume that the lysine-insensitive
aspartate kinase isoforms from Ltr*-1 and Ltr*-2 represent
different aspartate kinase isozymes. It is also possible that the lysine-sensitive
and lysine-insensitive subunits produced from alleles of the different
genes assemble into functional but lysine-insensitive heterodimers, based
on the lack of a detectable distinct anion exchange elution fraction with
the wildtype level of lysine sensitivity. Regardless of the mechanism involved,
relaxation of lysine feedback inhibition of corn aspartate kinase is associated
with overproduction of free threonine in vegetative and kernel tissues.
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