TY - JOUR

T1 - Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases

AU - Banerjee, Sanchari

AU - Muderspach, Sebastian J.

AU - Tandrup, Tobias

AU - Frandsen, Kristian Erik Høpfner

AU - Singh, Raushan K.

AU - Ipsen, Johan Orskov

AU - Hernandez-Rollan, Cristina

AU - Nørholm, Morten H. H.

AU - Bjerrum, Morten J.

AU - Johansen, Katja Salomon

AU - Lo Leggio, Leila

PY - 2022

Y1 - 2022

N2 - Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the N epsilon 2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

AB - Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the N epsilon 2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

KW - polysaccharide monooxygenase

KW - protonation

KW - stereochemical properties

KW - partial unrestrained refinement

KW - auxiliary activities

KW - SECONDARY COORDINATION SPHERE

KW - GLYCOSIDE HYDROLASE

KW - OXYGEN ACTIVATION

KW - PK(A) VALUES

KW - ACTIVE-SITE

KW - ENZYMES

KW - MECHANISMS

KW - CELLULOSE

KW - PROTEINS

KW - CLEAVAGE

U2 - 10.3390/biom12020194

DO - 10.3390/biom12020194

M3 - Journal article

C2 - 35204695

VL - 12

JO - Biomolecules

JF - Biomolecules

SN - 2218-273X

IS - 2

M1 - 194

ER -