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

Abstract

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 1 mm³ 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 2.15 Å 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 (Mg²⁺) or manganese (Mn²⁺) 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 Mn²⁺ and Mg²⁺ at the ion-binding site. This is supported by the different binding affinity of Mn²⁺ and Mg²⁺ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.

Keywords: Lactobacillus brevis; alcohol dehydrogenase; hydrogen-bonding network; neutron diffraction; protein crystallization; short-chain dehydrogenases/reductases.

Figure 1

LbADH-catalysed reduction of acetophenone (AC0) to (R)-1-phenyl­ethanol (S22).

Figure 2

Structural arrangement of LbADH in complex with NADP, acetophenone (AC0) and Mg²⁺ (PDB entry 1zk4). (a) Overall tetrameric arrangement of LbADH. The monomers are shown as differently coloured ribbon representations and the Mg²⁺ ion as a sphere; the NADP cofactor and AC0 substrate are highlighted as stick models (green and cyan, respectively). (b) Enlargement of the substrate and co-substrate binding region and (c) enlargement of the catalytic centre. This figure was prepared using PyMOL (v.2.1; Schrödinger).

Figure 3

Microscopic images of (a) the largest (∼1.0 mm³) and (b) the best neutron-diffracting His₆-tagged LbADH crystal (∼0.7 mm³).

Figure 4

Comparison of the unit cells of the neutron (PDB entry 6h1m; orange) and X-ray (PDB entry 6h07; green) structures. Monomers A of the X-ray and neutron structures were superimposed using the C^α atoms as a reference frame. Mg²⁺/Mn²⁺ ions in the neutron structure are coloured red and those in the X-ray structure are coloured blue. This figure was prepared using PyMOL (v.2.1; Schrödinger).

Figure 5

R.m.s. deviation between the neutron and X-ray structures for C^α atoms. This plot was prepared with MultiSeq (Roberts et al., 2006 ▸).

Figure 6

Mg²⁺/Mn²⁺-binding site coordinating four water molecules and connecting two C-terminal Gln251 residues of the biological unit. A difference OMIT map (F _o − DF _c) for both Gln251 residues, all water molecules and Mn²⁺/Mg²⁺ is contoured at 2.5σ. Red dashed lines indicate the coordination sphere for Mn²⁺/Mg²⁺. Distances are given in Å. (a) The nuclear scattering map for the neutron structure (orange; PDB entry 6h1m) shows no detectable density at the ion-binding site. Mn²⁺/Mg²⁺ was modelled in the neutron structure. (b) The electron scattering map for the X-ray structure (green; PDB entry 6h07) reveals clear density at the ion-binding site. This figure was prepared using CCP4mg (McNicholas et al., 2011 ▸).

Figure 7

Structural environment of the Mg²⁺/Mn²⁺ ion-binding site initiating a hydrogen-bonding pathway to Glu144. The neutron structure (orange; PDB entry 6h1m) and X-ray structure (green; PDB entry 6h07) are superimposed. A difference OMIT map (F _o − DF _c) for Wat6 of the neutron structure is contoured at 2.5σ. Blue dashed lines mark hydrogen bonds. Red dashed lines indicate the coordination sphere for Mn²⁺/Mg²⁺. Distances are given in Å and are measured according to the neutron structure. This figure was prepared using CCP4mg (McNicholas et al., 2011 ▸).

Figure 8

Influence of the Mn²⁺/Mg²⁺ ion-binding site on Lys210 in the neutron structure (orange; PDB entry 6h1m) and the X-ray structure (green; PDB entry 6h07). The hydrogen bonds are marked with blue dashed lines in the neutron structure. Black dashed lines in the X-ray structure mark the distances between O atoms. Red dashed lines indicate coordinated electrostatic bonds originating from Mn²⁺/Mg²⁺. Distances are given in Å. This figure was prepared using CCP4mg (McNicholas et al., 2011 ▸).