Jennifer A. McCourt

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

Summary.The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Thiamin nutrition and catalysis-induced instability of thiamin diphosphate

Thiamin (vitamin B1) is required in animal diets because it is the precursor of the enzyme cofactor, thiamin diphosphate. Unlike other B vitamins, the dietary thiamin requirement is proportional to non-fat energy intake but there is no obvious biochemical reason for this relationship. In the present communication we show for two enzymes that the cofactor undergoes a slow destruction during catalysis, which may explain the interdependence of thiamin and energy intakes.

Facile crystallization of Escherichia coli ketol-acid reductoisomerase.

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+ -dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP+, Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A resolution; attempts to phase them are currently in progress.