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Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis.
Biochemistry. 1996 Dec 17; 35(50):16282-91.B

Abstract

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and condensation of the resulting two-carbon moiety with a second alpha-keto acid. It belongs to a family of homologous, TPP-dependent enzymes which catalyze different reactions which start from decarboxylation of alpha-keto acids. A model for the structure of Escherichia coli AHAS isozyme II, based on its homology with pyruvate oxidase and experimental testing of the model by site-directed mutagenesis, has been used here to study how AHAS controls the chemical fate of a decarboxylated keto acid. Because of the potential conformational freedom of the reacting substrates, residues interacting with the substrate could not be identified directly from the model of AHAS. Three residues were considered as candidates for involvement in the recognition of alpha-ketobutyrate, as the amino acids at these sites in a unique low-specificity AHAS are different from those in typical AHASs, which are highly specific for reaction with alpha-ketobutyrate as second substrate, in preference to pyruvate. These residues were altered in AHAS II by site-directed mutagenesis. Replacement of Trp464 lowers the specificity by at least 1 order of magnitude, with minor effects on the activity or stability of the enzyme, suggesting that Trp464 contributes > or = 1.3 kcal mol-1 to interaction with the "extra" methyl of alpha-ketobutyrate. Mutations of Met460 or Thr70 have small effects on specificity and do affect other properties of the protein. A model for enzyme-substrate interactions can be proposed on the basis of these results. The model of AHAS also explains previously reported spontaneous mutants of AHAS resistant to sulfonylurea herbicides, which probably bind in the narrow depression which provides access to the bound TPP. A role for the C terminus of the enzyme polypeptide in determination on the reaction pathway is also possible.

Authors+Show Affiliations

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

8973202

Citation

Ibdah, M, et al. "Homology Modeling of the Structure of Bacterial Acetohydroxy Acid Synthase and Examination of the Active Site By Site-directed Mutagenesis." Biochemistry, vol. 35, no. 50, 1996, pp. 16282-91.
Ibdah M, Bar-Ilan A, Livnah O, et al. Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry. 1996;35(50):16282-91.
Ibdah, M., Bar-Ilan, A., Livnah, O., Schloss, J. V., Barak, Z., & Chipman, D. M. (1996). Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. Biochemistry, 35(50), 16282-91.
Ibdah M, et al. Homology Modeling of the Structure of Bacterial Acetohydroxy Acid Synthase and Examination of the Active Site By Site-directed Mutagenesis. Biochemistry. 1996 Dec 17;35(50):16282-91. PubMed PMID: 8973202.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis. AU - Ibdah,M, AU - Bar-Ilan,A, AU - Livnah,O, AU - Schloss,J V, AU - Barak,Z, AU - Chipman,D M, PY - 1996/12/17/pubmed PY - 1996/12/17/medline PY - 1996/12/17/entrez SP - 16282 EP - 91 JF - Biochemistry JO - Biochemistry VL - 35 IS - 50 N2 - Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and condensation of the resulting two-carbon moiety with a second alpha-keto acid. It belongs to a family of homologous, TPP-dependent enzymes which catalyze different reactions which start from decarboxylation of alpha-keto acids. A model for the structure of Escherichia coli AHAS isozyme II, based on its homology with pyruvate oxidase and experimental testing of the model by site-directed mutagenesis, has been used here to study how AHAS controls the chemical fate of a decarboxylated keto acid. Because of the potential conformational freedom of the reacting substrates, residues interacting with the substrate could not be identified directly from the model of AHAS. Three residues were considered as candidates for involvement in the recognition of alpha-ketobutyrate, as the amino acids at these sites in a unique low-specificity AHAS are different from those in typical AHASs, which are highly specific for reaction with alpha-ketobutyrate as second substrate, in preference to pyruvate. These residues were altered in AHAS II by site-directed mutagenesis. Replacement of Trp464 lowers the specificity by at least 1 order of magnitude, with minor effects on the activity or stability of the enzyme, suggesting that Trp464 contributes > or = 1.3 kcal mol-1 to interaction with the "extra" methyl of alpha-ketobutyrate. Mutations of Met460 or Thr70 have small effects on specificity and do affect other properties of the protein. A model for enzyme-substrate interactions can be proposed on the basis of these results. The model of AHAS also explains previously reported spontaneous mutants of AHAS resistant to sulfonylurea herbicides, which probably bind in the narrow depression which provides access to the bound TPP. A role for the C terminus of the enzyme polypeptide in determination on the reaction pathway is also possible. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8973202/Homology_modeling_of_the_structure_of_bacterial_acetohydroxy_acid_synthase_and_examination_of_the_active_site_by_site_directed_mutagenesis_ L2 - https://doi.org/10.1021/bi961588i DB - PRIME DP - Unbound Medicine ER -