Fig. 1: Multidomain structure of CbpD and evaluation of its activity on a model substrate.

a Schematic representation of the domain architecture of CbpD. The full-length protein contains a signal peptide (SP), an N-terminal AA10-type LPMO domain (AA10) followed by a module with unknown function (module X or MX) and a C-terminal CBM domain (CBM73). The residue boundaries for each domain, as well as potential post-translational modification (PTM) sites based on refs. ^25,35,36 and Supplementary Table 1, are labeled; phosphorylation sites are indicated above and lysine/arginine modifications below the domain illustration. PTMs identified in this study (Supplementary Table 1) are colored red. b Homology model of CbpD generated with Raptor-X⁹² showing flexible linkers in an extended conformation. The active site is indicated by a yellow partially transparent square. c SAXS model of monomeric CbpD (χ² = 2.35; produced with Pepsi-SAXS¹¹⁴; SASBDB ID: SASDK42), superimposed onto the ab initio SAXS model “envelope” (produced with DAMMIF^111,112 based on an average of 20 calculated models). Panels b and c were generated using PyMol. d Product formation by 1 µM of rCbpD_EC variants (FL: full-length; AA10: AA10 module only; MX + CBM73: the MX and CBM73 domains only) after a 2 h reaction at 37 °C with 10 mg ml⁻¹ β-chitin in 20 mM Tris-HCL pH 7.0, with 1 mM ascorbate as reducing agent, analyzed by HILIC. The degrees of polymerization (DP) of oxidized chitooligosaccharide aldonic acids in a standard sample are indicated. Control reactions without ascorbate did not show product formation. e HILIC analysis of reaction products emerging from a reaction of 1 µM rCbpD_EC with 10 mg ml⁻¹ β-chitin, 250 µM ascorbate in 20 mM Tris-HCl pH 7.0, and serial dilutions of pyocyanin (PCN) for 2 h at 37 °C. The chromatograms have been offset on the y axis to enable visual interpretation of their quantitative magnitude. Chromatograms for reactions containing serial dilutions of PCN and with or without various reductants that were sampled at different time points are shown in Supplementary Fig. 7c. f HILIC analysis of reaction products emerging from a reaction of 1 μM of copper-free full-length rCbpD_PA with 10 mg ml⁻¹ β-chitin in 20 mM Tris-HCL pH 7.0, 100 µM pyocyanin (PCN), 250 µM ascorbate, in the presence or absence of 1 µM azurin (Azu) after incubation for 2 h at 37 °C. Control reactions without added ascorbate did not show product formation (Supplementary Fig. 7d).