Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria

Abstract

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).

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

“…Further studies on expression profiling, gene inactivation and in vitro biochemical characterization of IlvB2, IlvG and IlvX will help to resolve these issues. Earlier studies on gene inactivation in enterobacteria have demonstrated that the multiplicity of AHAS has evolved to help the organisms in adapting to growth on C 2 or C 6 carbon sources, in the absence of external supplies of amino acids (Barak et al, 1987;Dailey et al, 1987). Since M. tuberculosis is known to utilize C 2 as the preferred carbon source for intracellular survival and replication (Muñoz-Elias & McKinney, 2006), it can be speculated that some AHAS isozymes might be functional under similar alternative physiological conditions.…”

“…AHAS has been well characterized in enterobacteria, and three isozymes of AHAS (type I IlvBN, type II IlvGM and type III IlvIH) have been reported from Escherichia coli and Salmonella typhimurium (Guardiola et al, 1974;Shaw et al, 1980). The three species of AHAS differ from each other in certain biochemical characteristics, such as sensitivity to valine and inhibition by sulfonylureas (Barak et al, 1987). In terms of their role in different physiological conditions, it has been postulated that the three isoforms of AHAS are critical for growth on different carbon sources, with AHAS I playing a major role during growth on acetate or palmitate, while AHAS II is required for growth in the presence of glucose (Barak et al, 1987;Dailey et al, 1987).…”

“…The three species of AHAS differ from each other in certain biochemical characteristics, such as sensitivity to valine and inhibition by sulfonylureas (Barak et al, 1987). In terms of their role in different physiological conditions, it has been postulated that the three isoforms of AHAS are critical for growth on different carbon sources, with AHAS I playing a major role during growth on acetate or palmitate, while AHAS II is required for growth in the presence of glucose (Barak et al, 1987;Dailey et al, 1987). It has been proposed that unlike Gram-negative bacteria, the Gram-positive bacteria possess a single species of AHAS.…”

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

“…2) for formation of lactyl-ThDP from bound pyruvate (kЈ 2), decarboxylation of lactyl-ThDP (kЈ 3), covalent ligation of the second ketoacid (kЈ 4), and release of AHA product (kЈ 5) were calculated by using Eqs. 6-9 from kcat and the distribution of ThDP-bound intermediates, determined by the rapid mixing-quench͞NMR method at 37°C in 0.1 M KPi (pH 7.6).…”

“…1) (5). Most AHASs show a strong preference for 2-ketobutyrate as the second substrate (6), which allows the pathway to function with an intracellular concentration of 2-ketobutyrate almost 2 orders of magnitude lower than that of pyruvate (7).…”