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Title: Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin

Here, lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding themore » mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [2] ;  [1] ;  [3] ;  [3] ;  [1] ;  [3] ;  [3] ;  [1] ;  [1] ;  [4] ;  [5]
  1. Univ. of Wisconsin, Madison, WI (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  5. Rice Univ., Houston, TX (United States)
Publication Date:
Grant/Contract Number:
AC02–05CH11231; AC02–06CH11357; P41GM103399; S10RR027000; GM109456; GM098248; AGM-12006; Grant 085P1000817; ACB-12002; AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 10; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; enzyme catalysis; enzyme mechanism; enzyme structure; lignin degradation; plant cell wall; protein structure; stereoselectivity; X-ray crystallography; structural enzymology
OSTI Identifier:
1257638
Alternate Identifier(s):
OSTI ID: 1379137

Helmich, Kate E., Pereira, Jose Henrique, Gall, Daniel L., Heins, Richard A., McAndrew, Ryan P., Bingman, Craig, Deng, Kai, Holland, Keefe C., Noguera, Daniel R., Simmons, Blake A., Sale, Kenneth L., Ralph, John, Donohue, Timothy J., Adams, Paul D., and Phillips, George N.. Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin. United States: N. p., Web. doi:10.1074/jbc.m115.694307.
Helmich, Kate E., Pereira, Jose Henrique, Gall, Daniel L., Heins, Richard A., McAndrew, Ryan P., Bingman, Craig, Deng, Kai, Holland, Keefe C., Noguera, Daniel R., Simmons, Blake A., Sale, Kenneth L., Ralph, John, Donohue, Timothy J., Adams, Paul D., & Phillips, George N.. Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin. United States. doi:10.1074/jbc.m115.694307.
Helmich, Kate E., Pereira, Jose Henrique, Gall, Daniel L., Heins, Richard A., McAndrew, Ryan P., Bingman, Craig, Deng, Kai, Holland, Keefe C., Noguera, Daniel R., Simmons, Blake A., Sale, Kenneth L., Ralph, John, Donohue, Timothy J., Adams, Paul D., and Phillips, George N.. 2015. "Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin". United States. doi:10.1074/jbc.m115.694307. https://www.osti.gov/servlets/purl/1257638.
@article{osti_1257638,
title = {Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin},
author = {Helmich, Kate E. and Pereira, Jose Henrique and Gall, Daniel L. and Heins, Richard A. and McAndrew, Ryan P. and Bingman, Craig and Deng, Kai and Holland, Keefe C. and Noguera, Daniel R. and Simmons, Blake A. and Sale, Kenneth L. and Ralph, John and Donohue, Timothy J. and Adams, Paul D. and Phillips, George N.},
abstractNote = {Here, lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.},
doi = {10.1074/jbc.m115.694307},
journal = {Journal of Biological Chemistry},
number = 10,
volume = 291,
place = {United States},
year = {2015},
month = {12}
}

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