Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase/protease I: a member of a new subclass of lipolytic enzymes.

Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20,500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H, 13C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four beta-strands and seven alpha-helices, arranged in alternate order. The four beta-strands were shown to form a parallel beta-sheet. The topological arrangement of the beta-strands of -1x, +2x, +1x appears to resemble that of the core region of the alpha beta hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of beta-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present.