Functional rescue of a bacterial dapA auxotroph with a plant cDNA library selects for mutant clones encoding a feedback-insensitive dihydrodipicolinate synthase.
Dihydrodipicolinate synthase (DHDPS; EC4.2.1.52) catalyses the first reaction of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes are strongly inhibited by lysine (I0.5 approximately 10 microM), whereas the bacterial enzymes are less (50-fold) or insensitive to lysine inhibition. We found that plant dhdps sequences expressing lysine-sensitive DHDPS enzymes are unable to complement a bacterial auxotroph, although a functional plant DHDPS enzyme is formed. As a consequence of this, plant dhdps cDNA clones which have been isolated through functional complementation using the DHDPS-deficient Escherichia coli strain encode mutated DHDPS enzymes impaired in lysine inhibition. The experiments outlined in this article emphasize that heterologous complementation can select for mutant clones when altered protein properties are requisite for functional rescue. In addition, the mutants rescued by heterologous complementation revealed a new critical amino acid substitution which renders lysine insensitivity to the plant DHDPS enzyme. An interpretation is given for the impaired inhibition mechanism of the mutant DHDPS enzyme by integrating the identified amino acid substitution in the DHDPS protein structure.
Vauterin
M
M
Laboratorium voor Plantengenetica, Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, Sint Genesius Rode B-1640, Belgium. mvauteri@vub.ac.be
Frankard
V
V
Jacobs
M
M
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005246:Feedback; D015723:Gene Library; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001051:Apoenzymes; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005020:Ethyl Methanesulfonate; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine