PMC

Stereo view (divergent, ‘wall-eyed’) of the putative substrate-binding faces of (a) Ao(AA13) depicting shallow groove leading through copper active site (N-terminal histidine shown in blue, copper ion shown in brown) and (b) a cellulose-active LPMO Ta(AA9) (Protein Data Bank code 3ZUD) with much flatter binding surface.

Release of maltose from retrograded starch by β-amylase over 4 h at 25 °C (columns 2–4) with An(AA13) (column 3), with reducing agent and An(AA13) (column 4). Maximum release of maltose was 36.8 nmol, which was 2.5 mol% of starch in the assay. Error bars represent s.e. of triplicate measurements. See Methods for more details.

MALDI–TOF–MS spectra of An(AA13) products from retrograded starch with 4 mM cysteine. C1-oxidized malto-oligosaccharides are present as a monosodiated lactone (m/z of the malto-oligosaccharide −2 Da), monosodiated aldonic acid (+16 Da) and disodiated aldonic acid (+38 Da). Inset: per-methylated C1-oxidized malto-oligosaccharides are present as monosodiated aldonic acid (+30 Da). The mass of unknown adduct X in peak e does not correspond to any cation component of the enzyme preparation.

Composite evolutionary tree with the AA13 part shown as a Newick-like cladogram for clarity and with the other LPMO families (AA9, AA10 and AA11, each with one representative sequence only for clarity) shown with branch lengths that represent phylogenetic distance. Bootstrap values are indicated for the two internal nodes separating the families. The two enzymes studied here are shown by An and Ao for the A. nidulans and the A. oryzae AA13 enzyme, respectively. AA13 sequences that are appended to a CBM20 module are shown in green, while those made only of an AA13 module are shown in black. The corresponding circular phylogram is presented in .

Structure of Ao(AA13). (a) Ribbon view of overall structure with numbered secondary structure elements, copper ion shown as orange sphere; (b) line diagram of active site; (c) stereo view of the electron density map around the active site in blue (contoured at 1.5σ) with anomalous difference density in yellow (contoured at 25σ), note methylation of N-terminal histidine (the map is calculated from the final refined structure with data collected at a wavelength of 1.037 Å); (d) comparison of active site of AA13 (green) with AA9 member (Protein Data Bank (PDB) 3ZUD, magenta, r.m.s.d. for protein atoms shown of 0.73 Å), AA10 member (PDB 2YOY, orange, r.m.s.d. of 0.53 Å) and AA11 member (PDB 4MAI, purple, r.m.s.d. of 0.60 Å).