--Janita M. Sellner, Robin A. Keith, Burle G. Gengenbach and David A. Somers
Dihydrodipicolinate synthase (DHPS) catalyzes the first enzymatic step specific to lysine biosynthesis. In maize, DHPS is feedback inhibited by lysine (I50 = 25uM). In previous studies (Frisch et al., Plant Physiol. 96:444, 1991; Frisch et al., Mol. Gen. Genet. 228:287, 1991), we purified maize DHPS to near homogeneity and isolated a corresponding full length cDNA. The maize cDNA was obtained by gene rescue in an Escherichia coli dapA- auxotroph that completely lacked DHPS activity. We have since used the dapA- strain AT997 to select for maize DHPS cDNAs with altered feedback inhibition properties.
Strain AT997 cells were transformed with the wildtype maize DHPS cDNA cloned into pUC119, mutagenized with EMS and grown on minimal medium such that growth depended on expression of maize DHPS. These cells were then subjected to selection for ability to grow in the presence of 10mM S-2-amino ethyl-L-cysteine (AEC), an analog of lysine that also inhibits maize DHPS activity. After 2 days, a number of colonies of putative AEC-resistant mutants were picked and rescreened for growth on AEC. Single cell isolates were grown and the isolated plasmids were then used to retransform auxotrophic AT997 cells to obtain clonal isolates of the mutant cDNAs.
To test whether the AEC-resistance of these colonies was conferred by a mutation in the maize DHPS gene that caused reduced feedback inhibition, DHPS activity in crude extracts was assayed in the presence of 10 to 1000uM lysine. Figure 1 shows data for two mutants that are representative of six maize DHPS cDNA mutants tested to date. In these tests, the expressed DHPS activity of the mutants was not inhibited by 1000uM lysine compared to >90% inhibition for wildtype maize DHPS at 100uM lysine. These data show a significant reduction in lysine sensitivity between the selected mutants and wildtype, and confirm that the bacterial auxotroph mutant selection protocol effectively identified lysine-resistant maize DHPS alleles. We currently are characterizing the DHPS activity from the DHPS mutant strains to determine the effect of the mutation on substrate kinetic properties of the enzyme.
Figure 1. Effect of increasing lysine concentrations on DHPS activity from E. coli cells expressing maize wildtype (DHPS+) or mutant (DHPS-m1 and DHPS-m2) DHPS. The wildtype E. coli DHPS activity is shown for comparison.
The DNA sequence of both strands of the DHPS-m1 cDNA clone conferring lysine-insensitive maize DHPS activity was determined by standard methods. Oligonucleotide primers used for sequencing were based on the sequence of the wildtype maize DHPS+ clone. Compared with the parental wildtype cDNA sequence (EMBL Data Bank accession no. 52850), only one nucleotide base change was present in the mutant cDNA region corresponding to the mature DHPS polypeptide. The mutation of C -> T occurred at position 497 resulting in the replacement of alanine (GCA) by valine (GTA) as shown:
wildtype DHPS cDNA ... 490 GTC CAC GCA ACA GAA 504 ...
Val His Ala Thr Glu
mutant DHPS cDNA ... 490 GTC CAC GTA ACA GAA 504 ...
Val His Val Thr Glu
A second maize cDNA isolate obtained after retransformation with the
original plasmid also had the same mutation. These comparisons indicate
that the single amino acid substitution can confer a high level of lysine
insensitivity to the mutant DHPS. Additional lysine-insensitive mutants
are being analyzed to fully resolve the nature of the lysine-recognition
site(s) of maize DHPS.
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