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Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

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Ketol‐acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched‐chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH‐dependent reduction of a 2‐ketoacid. The 2‐ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2‐ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2‐ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

We present in this paper a fast and stable global router called NTHU-Route 2.0 that improves the solution quality and runtime of a state-of-the-art router, NTHU-Route, by the following enhancements: (1) a new history based cost function, (2) new ordering methods for congested region identification and rip-up and reroute, and (3) two implementation techniques. The experimental results show that NTHU-Router 2.0 solves all ISPD98 benchmarks with very good quality. Moreover, it routes 7 of 8 ISPD07 benchmarks without any overflow. In particular, for one of the ISPD07 benchmarks which are thought to be difficult cases previously, NTHU-Route 2.0 can completely eliminate its total overflow. NTHU-Route 2.0 also successfully solves 12 of 16 ISPD08 benchmarks without causing any overflow.

The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans , attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K(i) value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell.

Resistance to sulfonylurea and imidazolinone herbicides can occur by mutations in acetohydroxyacid synthase (EC 4.1.3.18). Changing serine 653 to asparagine is known to cause insensitivity to imidazolinones but not to sulfonylureas. Here, S-653 of the Arabidopsis thaliana enzyme was mutated to alanine, threonine and phenylalanine. The purified mutated enzymes resemble wild-type in their enzymatic properties. The threonine and phenylalanine mutants are imidazolinone-resistant and the latter is also slightly sulfonylurea-resistant. The alanine mutant remains sensitive to both herbicides. The results suggest that the L L-hydroxyl group is not required for imidazolinone binding and that the size of the side-chain determines resistance.z 1999 Federation of European Biochemical Societies.

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