Functional expression of Nicotiana tabacum acetolactate synthase gene in Escherichia coli
Kim, H.J.; Chang, S.I.
Journal of Biochemistry and Molecular Biology 28(3): 265-270
1995
ISSN/ISBN: 1225-8687 Accession: 008709725
Acetolactate synthase (ALS, EC 4.1.3.18) is the first common enzyme in the biosynthesis of leucine, isoleucine, and valine. It is the target enzyme for several classes of herbicides, including the sulfonylureas, the imidazolinones, the triazolopyrimidines, the pyrimidyl-oxy-benzoates and the pyrimidyl-thio-benzens. The sulfonylurea-resistant ALS gene (SurB) from Nicotiana tabaccum (Lee et al. (1988) The EMBO J. 7, 1241-1248) was cloned into the bacterial expression plasmid pT7-7. The resulting recombinant plasmid pT7-ALS was used to transform an ALS-deficient Escherichia coli strain MF2000. MF2000 cells transformed with pT7-ALS grew in the absence of valine and isoleucine. ALS activities of 0.042 and 0.0002 mu-mol/min/mg protein were observed in the crude extracts prepared from MF2000 cells transformed with plasmids pT7-ALS and pT7-7, respectively. In addition, the former crude extract containing mutant ALS was insensitive to inhibition by K11570, a new chemical class of herbicides. IC-50 values for K11570 were 0.13 +- 0.01 mM. For comparison, a plasmid pTATX containing the wild-type Arabidopsis thaliana ALS coding sequences was also expressed in MF2000. ALS activities of 0.037 mu-mol/min/mg protein were observed, and the wild type ALS was sensitive to two different classes of herbicides, K11570 and ALLY, a sulfonylurea. IC-50 values for K11570 and ALLY were 0.63 +- 0.07 and 80 +- 5.6 nM, respectively. Thus, the results suggest that the sulfonylurea-resistant tobacco ALS was functionally expressed in the bacteria, and that K11570 herbicides bind to the regulatory site of ALS enzymes.