【For INTERNATIONAL COLLABORATIVE RESEARCH FY2023】

SUMMARY of

DATE: Day 07 Month 05 Year 2024

FY2023 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title

Crystal structure of glucose-6-phosphate 1-dehydrogenase

ApplicantName

Chun-Jung Chen

Affiliation

National Synchrotron Radiation Research Center

Present Title

Scientist / Professor

Research Collaborator (Host PI)

Prof. Atsushi Nakagawa

Summary

Glucose 6-phospate-1-dehydrogenase/6-phosphogluconolactonase (G6DP) is an important enzyme thatplays a major role in the pentose phosphate pathway (PPP). In the first step of PPP, glucose 6-phosphate isoxidized and NADP is reduced to produce 6-phosphoglucose lactone and NADPH, which is catalyzed by
G6DP. It is thus crucial to understand the structural and mechanistic roles of this enzyme. This project aims
to determine the crystal structures of G6DP at the highest resolution to elucidate the structural insights of theenzymatic mechanism. We performed crystal screening and collected X-ray diffraction data with a single
wavelength at the IPR BL44XU beamline of SPring-8. After several attempts, most of the G6DP crystals
diffracted X-ray to about 5 - 6 Å. We are currently optimizing the crystals and trying to improve the resolution
for complete structure determination in the future.
During this and previous proposal periods, we also collected several useful X-ray data sets ofendoglycosidase Sz and determined the various structures. Monoclonal antibodies (mAbs) have been
increasingly important and dominating in the drug markets for various human diseases and cancers.
Improvement of the therapeutic activities of mAbs has become a critical issue in the biomedical andbiopharmaceutical industry. We have discovered a new enzyme, EndoSz from Streptococcus equi subsp.
Zooepidemicus Sz105, and applied this novel enzyme to enhance the activities of mAbs with our developed
homogenous antibody platform. We demonstrate that the mutant EndoSz-D234M possesses an excellent
transglycosylation activity to generate diverse glycoconjugates on mAbs with homogeneous catalysis. We
also report the crystal structures of EndoSz-D234M in the apo-form and the complex form with the boundG2S2-oxazoline intermediate at high resolutions by utilizing a novel pH-jump method to improve resolution
significantly. Characterizations of transglycosylation activities with mutagenesis and crystal structures of
EndoSz-D234 in various forms with G2S2-oxazoline intermediate elucidate the transglycosylation
mechanism. Based on these findings, we develop a novel homogenous antibody platform with enhanced
therapeutic antibody efficacy that can greatly benefit the biomedical and pharmaceutical industries. The paper
titled “Structure-based high-efficiency homogeneous antibody platform by endoglycosidase Sz provides
insights into its transglycosylation mechanism” was published online at JACS Au,
https://doi.org/10.1021/jacsau.4c00004 on April 11, 2024.

*Deadline: May 10, 2024
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