RESUMO
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.
Assuntos
Acetolactato Sintase/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Cetol-Ácido Redutoisomerase/metabolismo , Subunidades Proteicas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/genética , Acetolactato Sintase/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Domínio Catalítico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Cetol-Ácido Redutoisomerase/química , Cetol-Ácido Redutoisomerase/genética , Peso Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Deleção de Sequência/genética , Valina/química , Valina/fisiologiaRESUMO
The enzyme acetohydroxyacid synthase (AHAS) catalyses the first common step in the biosynthesis of the three branched-chain amino acids. Enzymes in the AHAS family generally consist of regulatory and catalytic subunits. Here, we describe the first crystal structure of an AHAS regulatory subunit, the ilvH polypeptide, determined at a resolution of 1.75 A. IlvH is the regulatory subunit of one of three AHAS isozymes expressed in Escherichia coli, AHAS III. The protein is a dimer, with two beta alpha beta beta alpha beta ferredoxin domains in each monomer. The two N-terminal domains assemble to form an ACT domain structure remarkably close to the one predicted by us on the basis of the regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH). The two C-terminal domains combine so that their beta-sheets are roughly positioned back-to-back and perpendicular to the extended beta-sheet of the N-terminal ACT domain. On the basis of the properties of mutants and a comparison with 3PGDH, the effector (valine) binding sites can be located tentatively in two symmetrically related positions in the interface between a pair of N-terminal domains. The properties of mutants of the ilvH polypeptide outside the putative effector-binding site provide further insight into the functioning of the holoenzyme. The results of this study open avenues for further studies aimed at understanding the mechanism of regulation of AHAS by small-molecule effectors.
