SECONDARY STRUCTURE OF THE MUTT ENZYME AS DETERMINED BY NMR

The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the Halpha, C-13alpha, HN, N-15, and carbonyl C-13' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, Halpha and Calpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106. The antiparallel (a) or parallel (p) alignment of strands in the beta-sheet, based on 34 assigned long-range NOE peaks and 22 slowly exchanging amide NH protons, is C(a)D(p)A(a)B(a) E where strands C and D are connected by a type I tight turn, and strands A and B are connected by a nonclassical turn. Four loops (I, 24-46; II, 59-70; III, 88-101; and IV, 107-118) adopt an irregular, but well-defined structure with changes in direction via nonclassical turns occurring between residues 66-70, 88-90, and 113-115. All nine proline residues of MutT are trans, based on NOEs to preceding residues. Regions of flexibility, as judged by the absence of H-1-N-15 correlations in HSQC spectra and the absence of NOEs, are found at positions 25-31, 43, 112, and 118. Changes in chemical shifts and relaxation rates of N-15 and NH resonances occur in loop I in response to the binding of the activator Mg2+ and the substrate analogs Mg2+-AMPCPP and Mn2+-AMPCPP. Loop I also shows sequence identities with related enzymes from other bacteria suggesting this to be a portion of the active site.