Identification of point mutations which confer lysine-insensitivity to maize dihydrodipicolinate synthase
--Jonathan M. Shaver, Douglas C. Bittel, David A. Somers and Burle G. Gengenbach

Dihydrodipicolinate synthase (DHPS) catalyzes the first step specific to lysine biosynthesis in plants. DHPS is a key regulatory enzyme in the lysine biosynthetic pathway as it is sensitive to feedback inhibition by lysine. DHPS is also inhibited by a number of lysine analogs. The DHPS cDNA has been isolated from maize by selection for restoration of growth to an E. coli auxotroph DHPS- transformed with sequences from a maize cDNA library (Frisch et al., MGG, 1991). In an attempt to alter lysine regulation of DHPS, we have mutagenized the auxotrophic strain containing maize DHPS and selected for growth in the presence of a lysine analog, S-2-aminoethyl-L-cysteine.

From the time of the initial report in 1991 (Sellner et al., MNL 66:94, 1992), 12 additional mutants have been identified which are lysine-insensitive. Preliminary kinetic analyses of crude DHPS preparations have shown each mutant to be insensitive to 1 mM lysine compared to 50% inhibition at 25 uM for wildtype maize DHPS. No differences in sensitivity to lysine among the mutant forms of maize DHPS have been identified. The DHPS cDNA of six independently derived mutants has been sequenced resulting in the identification of three different mutations (see Figure 1).

The identification of four mutations within 10 residues of one another suggests that this particular region is an important domain for conferring lysine sensitivity to DHPS. This hypothesis is being tested by computer analysis of secondary structure of this region and by prediction of the changes caused by these mutations which alter the sensitivity to lysine. In addition, we are attempting to identify this region as a lysine-binding domain by comparison to other known lysine-binding proteins and by comparison to forms of DHPS from other species which are not inhibited by lysine.

Figure 1. Nucleotide sequence 458 to 508 and amino acid sequence 153 to 169 of maize DHPS are shown. The asterisk and boldface type represent the respective nucleotide and subsequent residue changes which confer lysine insensitivity to maize DHPS. The wildtype sequence is shown in the first block. Mutant 1-1 was described by Janita M. Sellner (MNL 66:94, 1992).


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