Solution structure and structural features of the parvulin NmPin from Nitrosopumilus maritimus. a Assigned 1H-15N-HSQC of recombinant NmPin. Assignment was performed with CcpNmr analysis using HNCACB/CBCACONH spectra to trace the protein sequence chain. b Ribbon presentation of the tertiary structure of NmPin. The parvulin fold comprises a central four-stranded β-sheet (red) surrounded by four α-helices (blue), which are connected through short loops and turns (cyan). Secondary structure elements are annotated with Greek letters. c Superposition of the structures of NmPin (blue) and Par14 (grey, PDB ID: 3UI4) with the two histidines (dual histidine motive ) in the respective leading color. The outer residues of the catalytic tetrad of NmPin (D42 in cyan, S82 in red) and of hPar14 (D74 and T118 in brackets)  are indicated. d A diagram of normalized isomerase activity measured for wildtype NmPin (black column) and proteins comprising either S82A (red column) or D42A (cyan column) single residue mutations within the catalytic tetrad. The mutants S82A and D42A retain only a low residual activity of 3.5 % and 0.7 %, respectively. Data were recorded in duplicates and are presented as means ± standard deviation. e Circular dichroism spectra of NmPin (black), NmPinD42A (cyan) and NmPinS82A (red) shown as mean residue ellipticity (mrw). *Datasets were normalized to the wildtype spectrum for better comparison of the three protein folds. f Surface representation of NmPin. Residues involved in substrate binding are mapped on the molecular surface (red, side chain atoms in purple sticks). Residues were derived from chemical shift perturbation analysis in a 1H-15N-SOFAST-HMQC titration experiment using Suc-A-R-P-F-pNA as a ligand. g Diagram of the normalized activity for various substrates comprising the scaffold Suc-A-X-P-F-pNA measured for NmPinD42C. The residue X is specified on the x-axis of the diagram. Data are normalized to the substrate with the highest activity and are presented as means ± standard deviation from two independent measurements with different enzyme concentrations. h and i Electrostatic potential of the molecular surface of NmPin and CsPin calculated with YASARA  using the Particle Mesh Ewald approach. The intensity of the surface potential is gradually colored from dark red (negative) over grey (neutral) to dark blue (positive) representing energy levels from –350 to +350 kJ/mol. The active site of NmPin is encircled. The opposite sites of both NmPin as well as CsPin are defined by a positively charged lysine-rich patch as labelled, respectively.