Dataset Information

Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.

ABSTRACT:

Background

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs.

Results

Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (^-Seg1) and a longer Segment 2 (⁺Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (⁺Seg1^-Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl-galactosyl and xylosyl-galactosyl-fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a ^-Seg1⁺Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the ⁺Seg1^-Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a ^-Seg1^-Seg2 configuration and no oxidative XG activity.

Conclusions

The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure-function relationship of AA9 LPMOs.

SUBMITTER: Sun P

PROVIDER: S-EPMC7257166 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO2]+ complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H2O2, which was recently suggested to be the true co-substrate, rather than O2. The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol-1, compared to the much higher barrier (156 kJ mol-1) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete to date and concur with available experimental evidence.