Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways

Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1mm ³ suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using the BIODIFF instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany to a resolution d _min of 2.15Å in 16 days. This allowed the first neutron crystal structure of LbADH to be determined. The neutron structure revealed new details of the hydrogen-bonding network originating from the ion-binding site of LbADH and provided new insights into the reasons why divalent magnesium (Mg ²⁺ ) or manganese (Mn ²⁺ ) ions are necessary for its activity. X-ray diffraction data were obtained from the same crystal at the European Synchrotron Radiation Facility (ESRF), Grenoble, France to a resolution d _min of 1.48Å. The high-resolution X-ray structure suggested partial occupancy of Mn ²⁺ and Mg ²⁺ at the ion-binding site. This is supported by the different binding affinity of Mn ²⁺ and Mg ²⁺ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.Large single crystals of the alcohol dehydrogenase from Lactobacillus brevis were generated, thus enabling neutron diffraction experiments to be performed for the first time with this protein. The obtained neutron structure revealed new details of the hydrogen-bonding network and provided new insights into the reasons why divalent magnesium or manganese ions are necessary for its activity.