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SUMMARY:Time-resolved serial crystallograhy to capture reaction intermedia
 tes of a glucuronyl esterase
DTSTART;VALUE=DATE-TIME:20250922T154000Z
DTEND;VALUE=DATE-TIME:20250922T155000Z
DTSTAMP;VALUE=DATE-TIME:20260526T060939Z
UID:indico-contribution-1833@lindico453.srv.lu.se
DESCRIPTION:Speakers: Gabrielle Wehlander (University of Gothenburg)\nGluc
 uronyl esterases (GEs) from the carbohydrate esterase family 15 (CE15) are
  involved in degrading lignocellulosic biomass\, by catalyzing the hydroly
 sis of an esterbond connecting lignin and hemicellulose in the plant cell 
 wall (1). In order to utilize biomass in biorefineries\, efficient methods
  are needed to separate cellulose\, hemicellulose and lignin. Studying GEs
  to better understand their reaction mechanism can aid in improving existi
 ng biological preteatment methods used in biorefineries to be able to make
  better use of this renewable energy source. The bacterial GE from Opitutu
 s terrae (OtCE15A) has previously been structurally determined at cryo-tem
 perature and a reaction mechanism for the acylation and deacylation reacti
 ons has been proposed (2\,3). Various glucuronate- and galacturonate ester
 s have been used as model substrates for the lignin-hemicellulose linkage\
 , and the substrates have been soaked into the crystals. However\, attempt
 s to capture the binding of the substrates prior to hydrolysis of the este
 r bond have so far been unsuccessful\, but a covalent reaction intermediat
 e has been obtained using enzymes with mutations at the catalytic site. In
  attempts to capture the binding of substrates prior to hydrolysis and to 
 determine reaction intermediates\, we have collected serial synchrotron X-
 ray crystallography (SSX) data at BioMAX (MAX IV\, Lund)\, and conducted i
 nitial time-resolved SSX experiment at P14.EH2 (T-REXX of PETRA III\, Hamb
 urg). We have obtained high resolution (1.7Å) SSX data of OtCE15A at BioM
 AX and observed binding of the cleaved substrate of benzyl glucuronoate af
 ter a soaking time of 5 minutes. For time-resolved SSX experiments at T-RE
 XX\, we have tested and are planning to use the hit-and-return (HARE) meth
 od (4).\n\nReferences\n1. Larsbrink\, Johan\, and Leila Lo Leggio. "Glucur
 onoyl esterases–enzymes to decouple lignin and carbohydrates and enable 
 better utilization of renewable plant biomass." Essays in Biochemistry 67.
 3 (2023): 493-503.\n2. Mazurkewich\, Scott\, et al. "Structural and bioche
 mical studies of the glucuronoyl esterase OtCE15A illuminate its interacti
 on with lignocellulosic components." Journal of Biological Chemistry 294.5
 2 (2019): 19978-19987.\n3. Zong\, Zhiyou\, et al. "Mechanism and biomass a
 ssociation of glucuronoyl esterase: an α/β hydrolase with potential in b
 iomass conversion." Nature Communications 13.1 (2022): 1449.\n4. Schulz\, 
 Eike C.\, et al. "The hit-and-return system enables efficient time-resolve
 d serial synchrotron crystallography." Nature methods 15.11 (2018): 901-90
 4.\n\nAuthors: Gabrielle Wehlander\, Josefin Ridaeus\, Scott Mazurkewich\,
  Leila Lo Leggio\, Johan Larsbrink\, Gisela Brändén\n\nhttps://lindico45
 3.srv.lu.se/event/583/contributions/1833/
LOCATION:LINXS at The Loop
URL:https://lindico453.srv.lu.se/event/583/contributions/1833/
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