4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: Backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

James T. Stivers, Chitrananda Abeygunawardana, Christian P. Whitman, Albert S. Mildvan

Research output: Contribution to journalArticlepeer-review

Abstract

4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated α-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an α-helix (residues 13-30), two β-strands (β1, residues 2-8; β2, residues 39-45), a β-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The β1 strand is parallel to the β2 strand of the same subunit on the basis of cross strand NHi-NHj NOEs in a 2D 15N-edited 1H-NOESY spectrum of hexameric 4-OT containing two 15N-labeled subunits/hexamer. The β1 strand is also antiparallel to another β1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the β-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.

Original languageEnglish (US)
Pages (from-to)729-741
Number of pages13
JournalProtein Science
Volume5
Issue number4
DOIs
StatePublished - Apr 1996

Keywords

  • Active site residues
  • NMR
  • Structure

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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