Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

Keywords: His-brace; N-terminal Arg-AA9; biomass degradation; copper monooxygenase; crystal structure; glycobiology; phosphorylation; polysaccharide; xylooligosaccharide.

Figure 1.

Schematic representation of oxidative cleavage of cellooligosaccharides/cellulose by LPMOs.

Figure 2.

AA9s with N-terminal Arg up-regulated on lignocellulosic biomass. Multiple sequence alignment of Arg-AA9 sequences found to be up-regulated during growth on aspen, pine, and wheat straw. The signal peptides are included for each sequence. The N-terminal Arg of the mature proteins is indicated with a star above the sequence. Numbering above the sequence alignment is according to the mature LsAA9B protein with Arg in the N terminus. Secondary structure elements of LsAA9B are shown above the alignment in yellow and red for β-strands and α-helices, respectively. The JGI protein ID is given next to each sequence. Residues with more than 70% sequence identity are indicated with blue (i.e. residues shared by at least seven of the nine protein sequences).

Figure 3.

Sequence coverage of LsAA9B peptides found in the L. similis secretome following growth on hardwood pulp.

Figure 4.

Structure of LsAA9B and positions equivalent to critical residues in AA9 LPMOs. a, side (left) and top (right) view of LsAA9B showing the common immunoglobulin-like β-sandwich-fold. b, detailed view with electron density near the N-terminal Arg (Arg-1), including phosphorylated Ser-25 (pS25). Ser-25 is modeled in two conformations (70 and 30% occupancies) with the highest occupied conformation being phosphorylated. The side chain of Arg-1 makes a 2.9 Å interaction with the phosphorylation on Ser-25. LsAA9B Arg-1, Asn-84, Leu-158, Gln-167, and Phe-169 are equivalent to His-1, His-68, His-142, Gln-151, and Tyr-153 in TtAA9E (residues critical for AA9 LPMO function). The electron density maps (2F_o − F_c in blue and F_o − F_c in green, contoured at 1.0σ and 3.0σ, respectively) are calculated from a structure before modeling the phosphorylation. The logo-conservation depict most common residues at a given position and their frequency (letter size) in Arg-AA9 sequences. c, active site residues of the structurally closest homolog NcAA9C (cyan, PDB 4D7U) superimposed onto the equivalent residues of LsAA9B.

Figure 5.

Surface features and conservation of LsAA9B. a, structure of LsAA9B interacting with a MES buffer molecule in a small pocket (magenta). The MES molecule makes an H-bond to His-65, and makes additional interactions with Tyr-71 and a GlcNAc molecule (N-linked glycosylation at Asn-134). b, electron density (2F_o − F_c (1.0σ) in blue and F_o − F_c (3.0σ) in green) for MES before modeling the buffer molecule. c, view of LsAA9B Ser-6, Asp-13, Gln-16, His-63, His-65, Tyr-71, and Asn-134-GlcNAc that form the pocket. d, residues of LsAA9B colored in a rainbow spectrum according to their conservation within Arg-AA9 sequences (red, completely conserved; yellow, intermediately conserved; blue, not conserved). Positions equivalent to residues involved in copper coordination and secondary coordination sphere in canonical AA9 LPMOs (Arg-1, Asn-84, Phe-169, Leu-158, Gln-167) and in substrate interactions in LsAA9A (Asn-26, His-66, Asn-67, Thr-83, Asn-159, Glu-161, Tyr-206) are shown as sticks. The speculated phosphorylation sites at Ser-25 and Ser-24 and the completely conserved residues in the L3 loop (His-65, Tyr-71, Pro-76, Pro-79, and Pro-82) are also shown in stick representation.

Figure 6.

Xylotetraose bound on the surface of LsAA9B. a and b, structure of LsAA9B-Xyl4 showing interaction with the xylotetraose ligand (Xyl4; yellow). The N-terminal Arg (Arg-1) and Asn-84 (equivalent to the His-brace of canonical AA9s) are shown in green. Positions involved in AA9-substrate interactions are colored magenta. The xylopentaose ligand (cyan) from the LsAA9A-Xyl5 complex structure (PDB 5NLO) are shown following superimposition onto LsAA9B. Blue surface indicates LsAA9B crystal contacts. The pSer-25 involved in crystal contacts are highlighted with an arrow. N-Linked glycosylation from a symmetry molecule (indicated with an asterisk) is found close to the Xyl5 ligand. c, electron density (2F_o − F_c (1.0σ) in blue and F_o − F_c (3.0σ) in green) calculated from a structure before modeling the Xyl4 ligand.