15
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Structural determinants of bacterial lytic polysaccharide monooxygenase functionality

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Bacterial lytic polysaccharide monooxygenases (LPMO10s) use redox chemistry to cleave glycosidic bonds in the two foremost recalcitrant polysaccharides found in nature, namely cellulose and chitin. Analysis of correlated mutations revealed that the substrate-binding and copper-containing surface of LPMO10s composes a network of co-evolved residues and interactions, whose roles in LPMO functionality are unclear. Here, we mutated a subset of these correlated residues in a newly characterized C1/C4-oxidizing LPMO10 from Micromonospora aurantiaca ( MaLPMO10B) to the corresponding residues in strictly C1-oxidizing LPMO10s. We found that surface properties near the catalytic copper, i.e. side chains likely to be involved in substrate positioning, are major determinants of the C1:C4 ratio. Several MaLPMO10B mutants almost completely lost C4-oxidizing activity while maintaining C1-oxidizing activity. These mutants also lost chitin-oxidizing activity, which is typically observed for C1/C4-oxidizing, but not for C1-oxidizing, cellulose-active LPMO10s. Selective loss in C1-oxidizing activity was not observed. Additional mutational experiments disclosed that neither truncation of the MaLPMO10B family 2 carbohydrate-binding module nor mutations altering access to the solvent-exposed axial copper coordination site significantly change the C1:C4 ratio. Importantly, several of the mutations that altered interactions with the substrate exhibited reduced stability. This effect could be explained by productive substrate binding that protects LPMOs from oxidative self-inactivation. We discuss these stability issues in view of recent findings on LPMO catalysis, such as the involvement of H 2O 2. Our results show that residues on the substrate-binding surface of LPMOs have co-evolved to optimize several of the interconnected properties: substrate binding and specificity, oxidative regioselectivity, catalytic efficiency, and stability.

          Related collections

          Author and article information

          Journal
          J Biol Chem
          J. Biol. Chem
          jbc
          jbc
          JBC
          The Journal of Biological Chemistry
          American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
          0021-9258
          1083-351X
          26 January 2018
          8 December 2017
          : 293
          : 4
          : 1397-1412
          Affiliations
          From the []Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway,
          [§ ]INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, and
          the []Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, 0318 Oslo, Norway, and
          the []Department of Microbiology, Clinic for Laboratory Medicine, Oslo University Hospital, Rikshospitalet, P. O. Box 4950, Nydalen, N-0424 Oslo, Norway
          Author notes
          [1 ] To whom correspondence should be addressed: Faculty of Chemistry, Biotechnology, and Food Science, The Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway. Tel.: 47-67232469; E-mail: zarah.forsberg@ 123456nmbu.no .

          Edited by Ruma Banerjee

          Article
          PMC5787815 PMC5787815 5787815 M117.817130
          10.1074/jbc.M117.817130
          5787815
          29222333
          92e79814-4109-491f-9b9b-4b25eb6d443a
          © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
          History
          : 11 September 2017
          : 22 November 2017
          Funding
          Funded by: Norges Forskningsråd , open-funder-registry 10.13039/501100005416;
          Award ID: 214138
          Award ID: 214613
          Award ID: 226247
          Funded by: The Norwegian academy of science and letters Vista programme
          Award ID: 6510
          Funded by: Marie-Curie FP7 COFUND People Programme
          Award ID: 267196
          Funded by: Helse Sør-Øst RHF , open-funder-registry 10.13039/501100006095;
          Award ID: 2015095
          Categories
          Enzymology

          site-directed mutagenesis,protein evolution,substrate specificity,regioselectivity,LPMO,chitin,hydrogen peroxide,cellulose

          Comments

          Comment on this article