Small molecules with big impact: fine static and dynamic detail in the atomic resolution structures of hydroxynitril lyase fromHevea brasiliensisgive insight into mechanisms of ligand binding

Abstract

Hydroxynitrile lyase (HNL) fromHevea brasiliensis is an enzyme of industrial use for the enantioselective production of cyanohydrins for organic synthesis from HCN and ketones. Previous X-ray crystallographic studies have helped to elucidate its reaction mechanism of HNL, yet some questions still remained unanswered. Although the catalytic reaction is base catalysed, the enzyme’s pH optimum lies within the pH region of 5 6. The pKa of the active site residues, in particular of those constituting the catalytic triad, is apparently adaptable subject to the conditions and the nature of the incoming ligands. Furthermore, the protonation state of adjacent residues relevant for ligand binding is yet unclear. Therefore, the effects of the ligands as well as the influence of the solvent pH on the protonation state in the active centre are the focus of the present study. The access to the HNL active site is very restricted; thus certain conformational changes must occur in order to admit the substrates and to release the products. We have developed a software tool that allows extracting directional motion of atoms, residues or groups of residues directly from the anisotropic atomic displacement parameters. In addition to various visualisation tools, the data include comprehensive statistics on the anisotropic behaviour within the protein and the assignment of regions of concerted motion. We collected a number of atomic resolution X-ray datasets on HNL in the native state as well as with soaked ligands such as acetone (one of the educts or products), isopropanol and thiocyanate. The structures were refined over the full resolution range including hydrogen atoms and anisotropic ADPs. The four crystal structures revealed the binding modes of the substrate analogues, which allowed the determination of the relative positions of the two co-substrates prior to cyanohydrin synthesis as well as monitoring changes in the arrangement and protonation state in the active site. Analysis of the anisotropic ADPs revealed differences in directional motion of the residues in the active site. In the cap region covering the active site the primary direction of motion changes relative to the hydrolase domain. The impact of ligand binding on the overall molecular motion as well as their correlation to fine structural details will be discussed.