4.7 Article

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity

期刊

出版社

MDPI
DOI: 10.3390/ijms20246219

关键词

enzyme engineering; lytic polysaccharide monooxygenase; phylogenetic analysis; regioselectivity; substrate binding; substrate specificity

资金

  1. European Union's Horizon 2020 research and innovation program (ERC Consolidator Grant OXIDISE) [726396]
  2. Doctoral Program Biomolecular Technology of Proteins (BioToP) - Austrian Science Fund (FWF) [W1224]

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In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

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