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13C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme–Substrate Complex of an Active, N-Linked Glycosylated Lipoxygenase

Biochemistry . . 2023-05; 
Ajay Sharma , Chris Whittington , Mohammed Jabed , S Gage Hill , Anastasiia Kostenko , Tao Yu , Pengfei Li , Peter E Doan , Brian M Hoffman , Adam R Offenbacher
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Gene Synthesis Gaeumannomyces graminis lipoxygenase, GgLOX (gene synthesized by Genscript), was expressed and purified in a similar manner to MoLOX. T/An endoglycosidase H (EndoH) gene from Streptomyces plicatus was synthesized by GenScript for recombinant expression in Escherichia coli and subcloned in line with his-tagged MBP construct (2CT-10 vector, Addgene plasmid #55209; gift from Scott Gradia). Get A Quote

Abstract

Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX-substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pa... More

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