The rates of formation of the two alternative products of acetohydroxy acid synthase (AHAS) have been determined by a new analytical method (N. Gollop, Z. Barak, and D. M. Chipman, Anal. Biochem., 160:323-331, 1987 (AHB) at approximately 180-and 60-fold faster rates, respectively, than acetolactate (AL) at equal pyruvate and 2-ketobutyrate concentrations. R values higher than 60 represent remarkably high specificity in favor of the substrate with one extra methylene group. In exponentially growing E. coli cells (under aerobic growth on glucose), which contain about 300 ,uM pyruvate and only 3 ,uM 2-ketobutyrate, AHAS I would produce almost entirely AL and only 1 to 2% AHB. However, isozymes II and III would synthesize AHB (on the pathway to Ile) and AL (on the pathway to valine-leucine) in essentially the ratio required for protein synthesis. The specificity ratio R of any AHAS isozyme was affected neither by the natural feedback inhibitors (Val, Ile) nor by the pH. On the basis of the specificities of the isozymes, the known regulation of AHAS I expression by the catabolite repression system, and the reported behavior of bacterial mutants containing single AHAS isozymes, we suggest that AHAS I enables a bacterium to cope with poor carbon sources, which lead to low endogenous pyruvate concentrations. Although AHAS II and III are well suited to producing the branched-chain amino acid precursors during growth on glucose, they would fail to provide appropriate quantities of AL when the concentration of pyruvate is relatively low.Acetohydroxy acid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase) catalyzes the condensation of an active acetaldehyde moiety derived from pyruvate with either another molecule of pyruvate, to form 2-acetolactate (AL), or with 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate (AHB) (28). The synthesis of the acetohydroxy acids is the first of a series of common steps in the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine ( Fig. 1) (5, 28, 29). The two AHAS reactions are the key steps in this pathway, as they are irreversible and committed steps toward the synthesis of two different sets of products; furthermore, the formation of AL is the first committed step on the valine pathway. There are several conceivable strategies for the regulation of such separate but interrelated biosynthetic pathways. (i) A single enzyme might operate with variable, metabolically controlled specificity for the two reactions (e.g., as in the case of ribonucleotide diphosphate reductase [27]). (ii) Different enzymes might specialize almost exclusively in one or another reaction, so that each could be separately controlled. (iii) The relative rates of the two reactions might be controlled by the relative concentrations of the precursors. The information available on AHAS has not been sufficient to determine to what extent each of these strategies has been adopted, even in the well-studied cases of Escherichia coli and Salmonella typhimurium (5, 29).

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial)hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.

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...

Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormnally high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria.

Acetohydroxy acid synthase (AHAS; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/2/1/6.html) is a thiamin diphosphate (ThDP)‐dependent decarboxylase‐ligase that catalyzes the first common step in the biosynthesis of branched‐chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl‐ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2‐ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2‐ketobutyrate (2‐KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing–quench NMR method or stopped‐flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2‐lactyl‐ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2‐KB, putting into question whether AHAS operates via a pure ping–pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor–acceptor:enzyme complex followed by covalent, ThDP‐promoted catalysis with concerted decarboxylation–carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.