Fig. 1: Structural and Biochemical Analyses of P. putida HglS.

a Ribbon diagram of the HglS crystal structure. The left image shows the active site entrance, containing the metal cofactor (red sphere) and metal-coordinating residues (orange) located in the central β-sheet protein domain. A 180° rotation on the right shows the overall protein structure. b Overlay of holo (cyan) and substrate-bound (green) structures of HglS displaying the enzyme active site and the ionic interaction between Arg74 and the distal carboxylate group of the 2OA substrate. The nickel bound within each structure is displayed as a red (holo structure) or purple (substrate-bound structure) sphere. c Proposed reaction mechanism of HglS. O₂-derived oxygens are shown in red. d Oxygen to 2OA stoichiometry of the HglS reaction. Dissolved oxygen concentration (y-axis) was measured with a Clark-type oxygen probe. Either 200 µM (red line) or 100 µM (blue line) of 2OA was added to initiate reaction, resulting in equimolar oxygen consumption. Dotted lines represent the expected final O₂ concentration when f100 µM (blue line) or 200 µM (red line) 2OA is consumed. e LC-HRMS extracted ion chromatograms (EICs) showing labeled ¹⁸O incorporation into the 2-hydroxyglutarate product of HglS. On the left (black lines) are EICs for ions with m/z 147.029, representing 2-hydroxyglutarate containing ¹⁶O. On the right (red lines) are EICs for ions with m/z 151.022, representing 2-hydroxyglutarate containing two ¹⁸O atoms. A control reaction performed under ambient O₂ conditions (n = 3) is compared with a reaction performed under a ¹⁸O₂ atmosphere (n = 3) and a 2-hydroxyglutarate standard. f Overlay of holo (green) and substrate-bound (cyan) structures of HglS displaying the enzyme active site and the interaction of Val402 and Ser403 with the 2-oxoadipate substrate. The loop containing Val402 and Ser403 is shown in the holo (dark green) and substrate-bound (dark blue) states. g Reaction rates of HglS with different 2-oxoacid substrates: 2-oxoglutarate (2OG), 2-oxoadipate (2OA), 2-oxopimelate (2OP), 2-oxobutyrate (2OB), 2-oxovalerate (2OV), 2-oxohexanoate (2OH), 2-oxooctanoate (2OO). Error bars represent 95% confidence intervals, black dots represent individual measurements, n = 3. h Reaction rates of WT HglS (black dashes), R74A (blue dashes), and V402P (orange dashes) mutants measured by an enzyme coupled decarboxylation assay with 2OA as a substrate, n = 3. Error bars represent 95% confidence intervals, colored dots correspond to individual measurements.