Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron.

Autor:	Zielinski KA; School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853., Dolamore C; Department of Biochemistry, University of Nebraska, Lincoln, NE 68588., Dalton KM; Department of Biology, New York University, New York, NY 10003.; Linac Coherent Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025.; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138., Smith N; Department of Biochemistry, University of Nebraska, Lincoln, NE 68588., Termini J; Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, CA 91010., Henning R; BioCARS, Center for Advanced Radiation Sources, The University of Chicago, Lemont, IL 60439., Srajer V; BioCARS, Center for Advanced Radiation Sources, The University of Chicago, Lemont, IL 60439., Hekstra DR; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.; John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138., Pollack L; School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853., Wilson MA; Department of Biochemistry, University of Nebraska, Lincoln, NE 68588.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Jul 20. Date of Electronic Publication: 2024 Jul 20.
DOI:	10.1101/2024.07.19.604369
Abstrakt:	DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.
Databáze:	MEDLINE
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