title: Substrate directs enzyme dynamics by bridging distal sites: UDP-galactopyranose mutase.
abstract: UDP-Galactopyranose mutase (UGM) is a flavoenzyme that catalyzes interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf); its activity depends on FAD redox state. The enzyme is vital to many pathogens, not native to mammals, and is an important drug target. We have probed binding of substrate, UDP-Galp, and UDP to wild type and W160A UGM from K. pneumoniae, and propose that substrate directs recognition loop dynamics by bridging distal FAD and W160 sites; W160 interacts with uracil of the substrate and is functionally essential. Enhanced Trp fluorescence upon substrate binding to UGM indicates conformational changes remote from the binding site because the fluorescence is unchanged upon binding to W70F/W290F UGM where W160 is the sole Trp. MD simulations map these changes to recognition loop closure to coordinate substrate. This requires galactose-FAD interactions as Trp fluorescence is unchanged upon substrate binding to oxidized UGM, or binding of UDP to either form of the enzyme, and MD show heightened recognition loop mobility in complexes with UDP. Consistent with substrate-directed loop closure, UDP binds 10-fold more tightly to oxidized UGM, yet substrate binds tighter to reduced UGM. This requires the W160-U interaction because redox-switched binding affinity of substrate reverses in the W160A mutant where it only binds when oxidized. Without the anchoring W160-U interaction, an alternative binding mode for UDP is detected, and STD-NMR experiments show simultaneous binding of UDP-Galp and UDP to different subsites in oxidized W160A UGM: Substrate no longer directs recognition loop dynamics to coordinate tight binding to the reduced enzyme.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Yao
authors.forename: Dustin W
authors.initials: DW
authors.lastname: Bleile
authors.forename: Yue
authors.initials: Y
authors.lastname: Yuan
authors.forename: Jay
authors.initials: J
authors.lastname: Chao
authors.forename: Karunan P
authors.initials: KP
authors.lastname: Sarathy
authors.forename: David A R
authors.initials: DA
authors.lastname: Sanders
authors.forename: B Mario
authors.initials: BM
authors.lastname: Pinto
authors.forename: Melanie A
authors.initials: MA
authors.lastname: O'Neill