Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Abstract

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.Lytic polysaccharide monooxygenases (LPMOs) 3 are enzymes that utilize copper as their functional active site metal and act to enhance the depolymerization of recalcitrant polysaccharides in nature such as cellulose and chitin (1, 2). Identification and characterization of members of this cuproenzyme family from fungal and bacterial species have gathered pace in recent years due in part to their promising uses in biorefinery applications of biomass to derive second generation biofuels (2, 3). LPMOs are classed into auxiliary activity (AA) families in the CAZy (carbohydrate active enzyme) database with four AA families, AA9, AA10, AA11, and AA13 identified so far (4). AA9 (formerly GH61) and AA10 (formerly CBM33) families have been the most extensively studied, with AA9 members having activity for cellulose, hemicellulose, and cellodextrin substrates (5-8) and AA10 members having activity with either cellulose or chitin substrates (9, 10). The AA11 and AA13 families are the most recently discovered, and so far have been determined to have activity with chitin and starch substrates, respectively (11, 12). All LPMOs coordinate a single copper ion by three nitrogen ligands to from the active site. Two of these are provided from the amino group (Nt) and side chain of the N-terminal His with the third from a second His side chain to giv...

Methylation of the N‐terminal histidine protects a lytic polysaccharide monooxygenase from auto‐oxidative inactivation

“…The slopes of the k obs versus [Cu 2+ ] plots yielded second‐order rate constants of 2.19 ± 0.06 × 10 6 and 1.77 ±0.04 × 10 6 M −1 ·s −1 , for Ta LPMO9A‐Ao and Ta LPMO9A‐Pp, respectively. Both linear plots cross the axes close to the origin, as previously observed in similar analysis of a family AA10 LPMO from Streptomyces lividans , Sli LPMO10E, suggesting that association rates for Cu 2+ are high compared with the dissociation rates …”

“…Both linear plots cross the axes close to the origin, as previously observed in similar analysis of a family AA10 LPMO from Streptomyces lividans, SliLP-MO10E, suggesting that association rates for Cu 2+ are high compared with the dissociation rates. 39 Redox potentials, assessed through equilibrium reactions with N,N,N 0 ,N 0 -tetramethyl-1,4-phenylenediamine (TMP), were determined to be 195 AE 7 mV for TaLPMO9A-Ao and 186 AE 5 mV for TaLP-MO9A-Pp.…”

Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

“…The ability of LPMOs to withstand EDTA has been reported before (8) and coincides with very high reported copper affinities (8,42). Apparently, the strong binding of copper and slow off rates (43) ensure that there is no significant transfer of copper from the LPMO to EDTA, provided that the contact time is kept short, as is the case here. Accordingly, in experiments with 0.1 mM AscA as reductant and 20 M load of H 2 O 2 , EDTA had no effect on the kinetics of SmLPMO10A.…”

Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens

“…This is not uncommon for these enzymes given their high affinity for copper which is very hard to remove from glassware and buffers and is scavenged rapidly by the enzyme, making it very challenging to completely demetallate LPMOs. This should not have much influence on the measured K D as seen here, as the obtained values match closely to those published for other enzymes in this family 31,32,34 .…”

“…The breadth of the peaks also indicated that there was potentially a mixture of species present in solution, as has recently shown by Chaplin et al 34 . As previously discussed, the reduced A z  value could arise from mixing of d(z 2 ) orbital Please do not adjust margins…”