Room-Temperature Structure of Xylitol-Bound Glucose Isomerase by Serial Crystallography: Xylitol Binding in the M1 Site Induces Release of Metal Bound in the M2 Site

Abstract

Glucose isomerase (GI) is an important enzyme that is widely used in industrial applications, such as in the production of high-fructose corn syrup or bioethanol. Studying inhibitor effects on GI is important to deciphering GI-specific molecular functions, as well as potential industrial applications. Analysis of the existing xylitol-bound GI structure revealed low metal occupancy at the M2 site; however, it remains unknown why this phenomenon occurs. This study reports the room-temperature structures of native and xylitol-bound GI from Streptomyces rubiginosus (SruGI) determined by serial millisecond crystallography. The M1 site of native SruGI exhibits distorted octahedral coordination; however, xylitol binding results in the M1 site exhibit geometrically stable octahedral coordination. This change results in the rearrangement of metal-binding residues for the M1 and M2 sites, the latter of which previously displayed distorted metal coordination, resulting in unstable coordination of Mg2+ at the M2 site and possibly explaining the inducement of low metal-binding affinity. These results enhance the understanding of the configuration of the xylitol-bound state of SruGI and provide insights into its future industrial application.

Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

“…Serial crystallography (SX) using an X-ray free electron laser or synchrotron X-ray is an emerging technique for determining room-temperature structures with minimal radiation damage 1 – 3 . These room-temperature structures exhibit more biologically relevant structural flexibility compared to traditional cryocrystallography, and can provide reliable structural information on radiation-sensitive proteins 4 – 6 . Moreover, the SX technique enables visualization of time-resolved structural dynamics in pump-probe studies using optical laser or diffusion-based approaches 7 – 9 .…”

Serial X-ray Crystallography

“…In SX, radiation damage can be minimized because intense XFEL or synchrotron X-rays are used to collect data by exposing the crystals to X-rays for a very short time and only once [4,[12][13][14][15]. Additionally, it is useful for understanding the function of molecules in terms of molecular dynamics because data collection is possible at room temperature or at near-physiological temperature [16,17]. Furthermore, by capturing structural changes at critical points in molecular activity through pump-probe experiments, it is possible to accurately distinguish molecular reaction mechanisms [18,19].…”

“…Using the SX technique, various room-or nearly physiological-temperature structures have been determined [16,17,[59][60][61]. This will provide useful information for understanding the flexibility of molecules when compared with cryogenic structures.…”

Molecular Dynamics—From Macromolecule to Small Molecules

“…In this Special Issue, we discuss molecular dynamics tools and related research results, covering small molecules to macromolecules. Specifically, we covered one editorial [ 1 ] and seven research articles [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ], as follows below.…”

“…Nam reported the room-temperature structure of glucose isomerase complexed with a xylitol inhibitor [ 4 ]. To provide more accurate structural information without radiation damage, serial synchrotron crystallography experiments were performed, and high-resolution crystal structures were determined.…”