Alternate conformations observed in catalytic serine of Bacillus subtilis lipase determined at 1.3 Å resolution

Bacillus subtilis extracellular lipase (BsL) has an exceptionally low molecular weight (19.4 kDa) for a member of the lipase family. A crystallographic study was performed on BsL in order to design and produce mutant BsL that will be more suitable for industrial uses based on analysis of the three-dimensional structure. Recently, the crystal structure of BsL has been determined at 1.5 Å resolution [van Pouderoyen et al. (2001). J. Mol. Biol. 309, 215–226]. In the present study, a new crystal form of BsL which provides diffraction data to higher resolution was obtained and its structure was determined at 1.3 Å using the MAD method. It was found that the active-site residue Ser77 has alternate side-chain conformations . The O^γ atom of the first conformer forms a hydrogen bond to the N^[epsilon] atom of His155, a member of the catalytic triad. In contrast, the second conformer is constructed with a hydrogen bond to the side-chain atom of the adjacent His76. These two conformers presumably correspond to the active and inactive states, respectively. Similar alternate conformations in the catalytic serine residue have been observed in Fusarium solani cutinase determined at 1.0 Å resolution and Penicillium purpurogenum acetylxylan esterase at 0.9 Å resolution. In addition, a glycerol molecule, which was used as a cryoprotectant, is found to be located in the active site. On the basis of these results, a model for substrate binding in the reaction-intermediate state of BsL is proposed.