A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity

Abstract

Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.

Structural and molecular dynamics studies of a C1‐oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

“…Extensive characterization studies of LPMOs encoded in fungal genomes have revealed that AA9 LPMOs vary in oxidization preference and secondary structure of the substrate-binding surface [18,19,26,27], which has attracted extensive research efforts to unveil structural insights into substrate recognition and catalytic mechanism of LPMOs [5, [32][33][34]49]. Adding to the current knowledge of the substrate interaction of C1specific LPMOs, we propose new insights and hypotheses for binding motifs and for the C1-specificity of HiLPMO9B.…”

“…However, elucidating LPMO–substrate interactions at the molecular level remains a significant challenge, as the most common AA9 LPMO substrate is insoluble cellulose . Accordingly, previous investigations of LPMO–substrate interactions have focused on LPMOs active on soluble polysaccharides .…”

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity

“…The cloning of Pc LPMO9D (Uniprot ID H1AE14), Nc LPMO9C (Q7SHI8), Hj LPMO9A (O14405), all with C‐terminal his 6 ‐tag, was described earlier . All mutants were created using Pfu Ultra High‐Fidelity DNA Polymerase (Agilent), the primers are listed in Tables S1–S4, Supporting Information.…”

“…After construct generation in E. coli BL21 (DE3), plasmid DNA was Swa I‐linearized and transformed into freshly prepared electro‐competent P. pastoris CBS7435_MutS cells . Selection of the best producing strain (typically out of 10 transformants) and shake flask LPMO production and purification was performed as previously described …”

“…The LPMO concentration in the ultrafiltrated culture broths was determined on SDS–PAGE gels using a BSA dilution series and ImageJ software, as described earlier …”