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Neutron and X‐ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen‐bonding pathways
Author(s) -
Hermann Johannes,
Nowotny Phillip,
Schrader Tobias E.,
Biggel Philipp,
Hekmat Dariusch,
Weuster-Botz Dirk
Publication year - 2018
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x18015273
Subject(s) - crystallography , neutron diffraction , chemistry , crystal structure , hydrogen bond , protein crystallization , crystallization , molecule , organic chemistry
Lactobacillus brevis alcohol dehydrogenase ( Lb ADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. Lb ADH 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 Lb ADH to generate large, single crystals with a volume of up to 1 mm 3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D‐exchanged Lb ADH crystal using the BIODIFF instrument at the Heinz Maier‐Leibnitz Zentrum (MLZ), Garching, Germany to a resolution d min of 2.15 Å in 16 days. This allowed the first neutron crystal structure of Lb ADH to be determined. The neutron structure revealed new details of the hydrogen‐bonding network originating from the ion‐binding site of Lb ADH 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 1.48 Å. 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.