Interactions between Large and Small Subunits of Different Acetohydroxyacid Synthase Isozymes of Escherichia coli

Abstract

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

“…The detailed feedback inhibition of AHAS is described below. A recent study based on the activation of each of the catalytic subunits with each of the regulatory subunits of E. coli isozymes compared the specificity of homologous (catalytic and regulatory subunits of same isozymes) and heterologous (catalytic and regulatory subunits of different isozymes) interactions [45]. It was found that the regulatory subunit of AHAS II can also activate the catalytic subunits of AHAS I and III, and that they also exhibit kinetic parameters similar to those observed in the enzymes reconstituted with the original catalytic and regulatory subunits.…”

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

“…This is in contrast to AHAS III [10] or, for example, the single AHASs of Mycobacterium avium [18] or Bacillus stearothermophilus [19], which all show noncompetitive partial inhibition by valine. The regulatory subunit (RSU) of AHAS I (ilvN) is a short protein of 96 amino acid residues (half the size of ilvH and other typical bacterial RSUs) [9,20,21].…”

Arginine 26 and Aspartic Acid 69 of the Regulatory Subunit are Key Residues of Subunits Interaction of Acetohydroxyacid Synthase Isozyme III from E. coli

“…17 Heterologous interaction has been detected between E. coli AHAS I and AHAS III in our previous study 18. Furthermore, ilvM (RSU of E. coli AHAS II) is able to activate the CSUs of all three AHAS isozymes from E. coli , and truncated ilvH (AHAS III) can activate the CSUs of both AHAS I and AHAS III 19. These results imply that there might be a common contact surface in the three isozymes of AHASs from E. coli.…”