The thiamin diphosphate (ThDP)-dependent biosynthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP ؊ ), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP ؊ from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP ؊ is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg 276 ) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound Acetohydroxyacid synthase (AHAS) 1 belongs to a homologous family of thiamin diphosphate (ThDP)-dependent enzymes that catalyze reactions whose initial step is decarboxylation of pyruvate or another 2-ketoacid (1, 2). However, despite the similarity of AHASs to, for example, pyruvate decarboxylases and pyruvate oxidases (3-6), AHASs carry out a specific carboligation reaction in which the decarboxylation of pyruvate is followed by the condensation of the bound hydroxyethyl-ThDP anion/enamine (HEThDP Ϫ ) intermediate with a second aliphatic ketoacid to form an acetohydroxyacid (Fig. 1). Whereas the role of the enzyme in the first steps in AHAS catalysis (i.e. activation of ThDP (7), decarboxylation of pyruvate, and formation of HEThDP Ϫ (step 1 in Fig. 1)) is comparable with the function of other members of its homologous family (8), it has been difficult to suggest roles for specific protein residues in the final steps (2 and 3) of the reaction in which the product acetohydroxyacid is formed and released.One reason for this uncertainty has been the lack of clear direct information on the structure of the regions of the active site that might be involved in selective reaction of HEThDP Ϫ with a second ketoacid. Although we have proposed a homology model for AHAS isozyme II from Escherichia coli (9), based on the crystal structure of pyruvate oxidase from Lactobacillus plantarum (LpPOX) (4), these two proteins have very different sequences in the region that is likely to interact with the second substrate. In the first published crystal structure of an AHAS, that of the catalytic subunits of the yeast enzyme (10), this region was disordered. The recent publication of a new structure of the yeast enzyme with a tightly bound herbicide (11) now provides a solid framework for consideration of the role of the protein in directing the fate of the HEThDP Ϫ intermediate. A second, equally serious obstacle to the understanding of the mechanism of AHAS has been a lack of experimental tools for studying the rates of individual steps in the reaction ...

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Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced. Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized. This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B. stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria.The first step common to the biosynthesis of branchedchain amino acids, catalyzed by acetohydroxyacid synthase (AHAS) (EC 4.1.3.18), is the condensation of pyruvate with either 2-ketobutyrate (the precursor of isoleucine) or pyruvate (the precursor of valine) (4, 26). Bacterial AHASs are composed of large (60-kDa) catalytic and small (9-to 18-kDa) regulatory subunits. Isolated catalytic subunits have lower activity than the holoenzymes but are similar to them in their cofactor dependence and specificity towards the two different substrates (10,27,28). The sensitivity of AHAS to feedback inhibition is completely dependent on the small subunit.Many bacteria and archaea apparently contain a single AHAS enzyme. In most gram-positive bacteria, the genes for the first two enzymes in the pathway are located in the same operon (ilvBNC) (5,9,13,15,30), often together with the leu genes (ilvBNC-leuACBD) (17,25,30). The enterobacteria contain three isozymes of AHAS, encoded by distinct and differently regulated operons (3, 4).To investigate the AHAS of Bacillus stearothermophilus (AHAS Bst ), we cloned the genes for this holoenzyme (ilvBN) and its large (ilvB) and small (ilvN) subunits to allow sequencing and overexpression. The screening for these genes was conducted with a genomic cosmid library for B. stearothermophilus ATCC 7954, created by H. Ewis (unpublished data), with a digitonin-labeled 1,100-bp probe that is highly conserved (50 to 75% amino acid identity) among AHASs (7) and only slightly conserved in other thiamine diphosphate (ThDP)-dependent enzymes, such as pyruvate oxidase (30%) and catabolic acetolactate synthase (25%) of Bacillus subtilis. This probe was amplified from the B. stearothermophilus ATCC 12980 genome by using two degenerate oligonucleotide primers: 5Ј(C/T/A)GGNACNGA(T/C)GCNTT(T/C)CA(A/G)GA and 5Ј T(C/G)(C/T)TGCCA(C/T)(T/G)NACCAT.The gene order in the insert of the AHAS-positive cosmid, as determined by coding analysis of its sequence (Fig. 1), seems similar to that of the B. subtilis ilv-leu operon (16, 30). The 5Ј end of ilvB was absent in the cosmid-cloned fragment. This region was added to the clone, as shown in Fig. 1, from a PCR-amplified fragment obtained from the genome of B. stearothermophilus ATCC 7954 by using primers that were identical to the T-box element of the ilv-leu operon from B. subtilis (14) (GGGTGGTACCGCGG) and to a sequenced 3Ј region of ilvB from B. stearothermophilus (GGCGGATTTGC CAATGGTTCGGC).The DNA sequences of the ilv-leu operon of B. stearothermophilus (NCBI accession no. AY083837) and the deduced...

The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.