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FIG. 1.
Traditional metabolite conversion pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. The enzymes involved in the common pathway for branched chain amino acid biosynthesis are TDA (EC 4.3.1.19), AHAS (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86), dihydroxy acid dehydrase (DAD) (EC 4.2.1.9), TB (EC 2.6.1.42), transaminase C (TC) (EC 2.6.1.66), 
-isopropylmalate synthase (IPMS) (EC 4.1.3.12), -isopropylmalate isomerase (IPMI) (EC 4.2.1.33), 
-isopropylmalate dehydrogenase (IPMDH) (EC 1.1.1.85), the L-leucine, L-isoleucine, and L-valine transporter I (LIV-I), and the L-leucine-specific (LS) transporter. The metabolites used were L-threonine (Thr), L-isoleucine (Ile), L-valine (Val), L-leucine (Leu), L-glutamate (Glu), Ala, Pyr, KB, -acetolactate (AL), -acetohydroxybutyrate (AHB), ,-dihydroxy-isovalerate (DHIV), ,-dihydroxy--methylvalerate (DMV), -ketoisovalerate (KIV), -keto--methylvalerate (KMV), -ketoglutarate (KG), -isopropylmalate (IPM), -isopropylmalate (IPM), -ketoisocaproate (KIC), extracellular L-isoleucine (ex-Ile), extracellular L-valine (ex-Val), and extracellular L-leucine (ex-Leu). The italicized items in parentheses refer to abbreviations used in the figure that are not defined in the Abbreviations footnote. Gene names for each enzyme are italicized in the figure. Enzyme reactions are indicated by arrows. Feedback inhibition patterns are indicated by dashed lines. Activation is indicated by a plus sign, and inhibitions are indicated by vertical bars. The line through AHAS II, ilvGM, indicates that this isozyme is not active in E. coli strain K12.
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FIG. 2.
Enzyme-centric, metabolic pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. The abbreviations of enzymes and metabolites are the same as those in Fig. 1. Ovals represent enzyme molecules. White ovals indicate free enzyme states, and shaded ovals indicate intermediate enzyme states with a function group attached to enzymes. Enzyme reactions are indicated by lines with arrowheads. Reversible reactions are indicated by gray lines with arrowheads. Switching between free and intermediate enzyme states are indicated by dashed lines with double arrowheads.
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FIG. 3.
Simulated flow of carbon through the branched chain amino acid biosynthetic pathways of E. coli K12. The graphical insets show the approach (minutes) to steady-state (µM) synthesis and utilization of the substrates, intermediates, and end products of the pathways. The intermediates are abbreviated as described in the legend of Fig. 1. The starting substrates L-threonine and pyruvate are supplied at rates to maintain constant levels of 520 and 1000 µM, respectively. L-Glutamate (Glu) and Ala for the transamination reactions are supplied at a rate to maintain constant levels of 2000 µM each. For the acetohydroxy acid isomeroreductase reaction, NADPH is supplied at a rate to maintain a constant level of 1000 µM. For the -isopropylmalate synthase (IPMS) reaction, acetyl-CoA is supplied at a rate to maintain a constant level of 1000 µM. The beginning substrates (L-threonine and pyruvate) levels, as well as the end product (L-isoleucine, L-valine, and L-leucine) levels, agree with measured intracellular values (21, 22). Where available, the ranges of reported values for pathway intermediate and end product levels in cells growing in a glucose minimal salts medium are shown in parentheses (µM) in the inset graphs.
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FIG. 4.
Allosteric regulation of L TDA. A, the fraction of TDA in the active R state. At t = 0 and an initial L-threonine concentration of 520 µM, 
65% of the TDA enzyme is in the active R state. As L-isoleucine accumulates, TDA is rapidly end product-inhibited and, as L-valine accumulates, this inhibition is slowly countered until, at steady state, only 5.5% of the total enzyme is in the active R state. B, the fractional saturation of TDA with L-threonine (vo/Vmax). At t = 0 and an initial L-threonine (Yf) concentration of 520 µM, 8% of the total enzyme is saturated with L-threonine. At a final steady-state level of end product synthesis, it is only 1.2% saturated with L-threonine.
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FIG. 5.
Simulated effects of excess L-valine on branched chain amino acid biosynthesis in E. coli K12. Conditions described in Fig. 2 were used for the simulations presented here, except that excess extra-cellular L-valine was added at a rate sufficient to be maintained at a concentration of 1 mM. The data in panel A show that, as described under "Results," excess L-valine increases rather than inhibits L-isoleucine biosynthesis. The data in panel B show that excess L-valine also causes a 4-fold increase in the intracellular accumulation of KB, which is restored to control levels by the extracellular addition of 500 µM L-isoleucine. The data in panel C show that the accumulation of KB observed in the presence of excess L-valine coincides with the conversion of nearly 18% of the cellular TDA to a catalytically active R state and that the subsequent extracellular addition of 500 µM L-isoleucine reverses this transition to the control level (Fig. 3A).
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FIG. 6.
Simulation of an E. coli K12 strain that overproduces L-isoleucine. The simulation conditions described in the Fig. 2 legend were used for the simulations presented here, except that a L-threonine deaminase feedback-resistant mutant, TDAR, was simulated by increasing its Ki for L-isoleucine to 100,000 µM, and the ilvGMEDA operon attenuator mutant (ilvGMEDA-att–) was simulated by increasing TDA, AHAS II, acetohydroxy acid isomeroreductase, dihydroxy acid dehydrase, and TB total enzyme levels 11-fold (26) (3). The simulation in panel A shows that the effect of the feedback-resistant TDA mutant TDAR is to allow the positive effector ligands L-threonine and L-valine to transition nearly 100% of the TDA enzyme to the active R state. The simulation results in panel B show that L-isoleucine production in the TDAR mutant is 5–6-fold increased. The simulation in panel C demonstrates that in the TDAR K12 mutant, the intermediate, KB, accumulates to a level 40-fold higher than in a wild type K12 strain; however, when the AHAS II isozyme is restored and the bi-functional enzymes of the L-isoleucine and L-valine pathways are genetically de-repressed 11-fold (ilvGMEDA-att–), KB accumulation is relieved (panel C) and L-isoleucine synthesis is increased more than 40-fold over the wild-type K12 level (panel D).
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FIG. 7.
An acetohydroxy acid isomeroreductase mutant (ilvC) E. coli K12 strain is auxotrophic for L-isoleucine and L-valine but not for L-leucine. The simulation conditions described in Fig. 2 were used for the simulations presented here, except that the initial concentration of acetohydroxy acid isomeroreductase was set to 0 to simulate an ilvC mutation, and extracellular L-valine and L-isoleucine were supplied at a level of 500 µM each. The results show that -ketoisovalerate (KIV) (panel A) and L-leucine (panel B) are produced in an ilvC strain in the presence of extracellular L-valine and L-isoleucine.
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