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Figure 2
Structure and biochemical study of MtEno. (a) Biochemical study of MtEno. (Top) Size-exclusion chromatogram of MtEno indicates that the protein exists in both octameric and dimeric forms in solution. The octameric fraction was determined to be more pure than the dimeric fraction using SDS–PAGE. (Bottom-left) The homogeneity and size of the MtEno octameric fraction were further confirmed through SEC–MALS. (Bottom-right) Michalis–Menten kinetics with Km = 495.7 ± 45.27 µM for the reversible activity of octameric enolase against PEP, and with no measurable activity for dimeric enolase against PEP (n = 2 for technical duplicates). (b) Biological assembly of MtEno in the apo state (XRD structure shown). (Top) Conventional dimeric arrangement is highlighted in red and the IF1 and IF2 interfaces are marked with dashed lines; highlighted dimer is shown in cartoon (middle) with twofold rotational symmetry marked. (Bottom) Monomer architecture demarcated with N-terminal (light blue) and C-terminal (grayscale and cyan) domains. β-Strands near the catalytic core are labeled by their order in the polypeptide chain from N- to C-termini. β-5 is antiparallel to the rest of the barrel and hence is colored differently. (c) Consurf analysis of enolase monomer (Ashkenazy et al., 2016BB901). Structures involved in forming the IF2 (left), the C-terminal β-barrel (middle) or the IF1 (right) are shown with faded a structure of the rest of the monomer in the background. Red and teal indicate the most and least conserved, respectively.

IUCrJ
Volume 10| Part 6| November 2023| Pages 738-753
ISSN: 2052-2525
https://doi.org/10.1107/S2052252523008485