Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

Abstract

Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μML-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from theAsk andAsk2 structural genes.

Molecular Genetics of the Maize (Zea mays L.) Aspartate Kinase-Homoserine Dehydrogenase Gene Family

“…Our analyses indicated that two ak-hsdh genes were genetically distinct from the askl and ask2 genes that encode Lys-sensitive AK. Dominant mutations in the ask genes cause AK to become Lys insensitive, which leads to Thr overproduction in maize kernels (Dotson et al, 1990a). askl is located on chromosome 7S (Azevedo et al, 1990), genetically distinct from ak-hsdh map locations on chromosome 2L and 4S.…”

“…Giovanelli et al (1989aGiovanelli et al ( , 1989b have shown that Lys and Thr feedback inhibition of AK may not be the major factor limiting Thr synthesis in Lemna paucicostata. However, the maize (Dotson et al, 1990a), barley (Hordeum vulgare) (Bright et al, 1982a(Bright et al, , 1982b, and tobacco (Nicotiana sylvestris) (Frankard et al, 1991) Ask mutants (Lys-insensitive AK mutants) and the transgenic tobacco plants containing a Lys-insensitive AK from E. coli (Shaul and Galili, 1992;Karchi et al, 1993) all exhibited the Thr-overproducing phenotype. These comparisons of mutant and wild-type plants provide compelling evidence for in vivo regulation of Thr synthesis in wild-type versions of these species.…”

Isolation and Characterization of Dihydrodipicolinate Synthase from Maize

“…Plants of inbred line A619 and two homozygous mutant lines, Ask-LTJ9 (12) and Ask2-LT20 (4), were grown in the field nursery at St. Paul, MN, in 1989. The Ask-LTJ9 and Ask2-LT20 alleles are altered forms of the aspartate kinase structural genes (Ask and Ask2) which result in reduced feedback inhibition of aspartate kinase by lysine (7). The two or three youngest, fully expanded leaves were harvested from plants at the five-leaf stage.…”

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

“…and of deregulated mutant plant enzymes (Dotson et al, 1990;Frankard et al, 1992) was helpful in elucidating the regulatory hierarchies. In vivo expression of bacterial enzymes with regulatory properties different from those of the corresponding plant enzymes allowed the analysis of effects of changes of the content of single amino acids on plant growth (Shaul and Galili, 1992;Karchi et al, 1993).…”