Functional expression of plant acetolactate synthase genes in Escherichia coli

Abstract

Acetolactate synthase (ALS; EC 4.1.3.18) is the first common enzyme in the biosynthetic pathways leading to leucine, isoleucine, and valine. It is the target enzyme for three classes of structurally unrelated herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. A cloned ALS gene from the small cruciferous plant Arabidopsis thaliana has been fused to bacterial transcription/translation signals and the resulting plasmid has been used to transform Escherichia coli. The cloned plant gene, which includes sequences encoding the chloroplast transit peptide, is functionally expressed in the bacteria. It is able to complement genetically a strain ofE. coli that lacks endogenous ALS activity. An ALS gene cloned from a line of Arabidopsis previously shown to be resistant to sulfonylurea herbicides has been similarly expressed in E. coli. The herbicide-resistance phenotype is expressed in the bacteria, as assayed by both enzyme activity and the ability to grow in the presence of herbicides. This system has been useful for purifying substantial amounts of the plant enzyme, for studying the sequence parameters involved in subcellular protein localization, and for characterizing the interactions that occur between ALS and its various inhibitors.

Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase

“…Additional removal of N-terminal amino acids beyond Thr-86 up to and including Asp-101 (replaced with the sequence Met-Gly) yields an inactive enzyme as seen with pTrc99A-AHAS : I102. Previously reported values for the specific activity of Arabidopsis AHAS (expressed from the intact gene) in the soluble fraction of cell lysate range from 0n037 unit\mg to 0n085 unit\mg [19][20][21]. Thus our values are comparable with those reported.…”

“…So far the enzyme has been obtained in apparently pure form from barley [17] and wheat leaves [18] only. AHAS from Arabidopsis thaliana [19][20][21], tobacco [21] and oilseed rape [22] has been functionally expressed in bacteria but the enzyme was not purified. More recently, Arabidopsis AHAS has been expressed in E. coli and purified as a glutathione S-transferase (GST) fusion protein.…”

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors

“…On the other hand, cells transformed with pAC210 (designated PA10) grew normally in the absence of valine and isoleucine. These results are similar to functional complementation of a microbe lacking the endogenous AHAS activity using the plant enzyme demonstrated before (20,23). To confirm our complementation observation, cells were grown in liquid culture in the presence or absence of valine and isoleucine.…”

“…The E. coli AHAS I requires a second subunit for sensitivity to valine inhibition (3). It has been shown that the AHAS gene from plants (without the transit peptide) and from E. coli are of similar size as well as they have high sequence homology (1,20). Therefore, it is possible that, similar to the E. coli enzyme, the plant enzyme also requires a second subunit for the feedback sensitivity.…”

“…Expression of enzymes in heterologous systems, such as in E. coli, has been successfully used in many cases to obtain large amounts of proteins for such studies. AHAS from Arabidopsis and Brassica napus have been expressed in E. coli and S. typhimurium, respectively (20,23). In these cases, functional complementation of growth of the microbes and sensitivity of the enzyme and the microbes to AHAS inhibitors were examined.…”