Regulation of the activity of a tetra-modular lytic polysaccharide monooxygenase from Enterobacter cloacae by inter-domain copper coordination

Enterobacter cloacae is a Gram-negative nosocomial human pathogen that inhabits diverse ecological niches. Its genome encodes a conserved set of putative chitin-active enzymes, including a lytic polysaccharide monooxygenase (LPMO), termed EcLPMO, which we functionally characterized in this study. EcLPMO is a tetra-modular protein consisting of an auxiliary activity family 10 (AA10) catalytic domain, two central domains of unknown function (DUF-A and DUF-B), and a C-terminal carbohydrate-binding module (CBM73). Functional assays using full-length EcLPMO and its truncated variants demonstrated that the AA10 domain oxidatively cleaves chitin at the C1 position. The CBM73 module enhances chitin binding and promotes synergy with endogenous chitinases. EcLPMO exhibited synergy with the unimodular chitinase EcChiA, resulting in up to 14-fold and 60-fold increase in GlcNAc release from α- and β-chitin, respectively. Deletion of both DUFs reduced EcLPMO activity. While DUF-A alone and the association of DUF-A and DUF-B showed limited chitin binding, DUF-B alone exhibited no binding, suggesting a distinct role. Using AlphaFold3, we observed that the DUF-B domain contains two highly conserved histidines that coordinate the AA10-bound copper, forming a previously unreported "inter-domain tetra-histidine copper coordination" center. These findings highlight the structural and functional complexity of EcLPMO and suggest that its accessory domains, particularly DUF-B, may contribute to enzyme stability and substrate interaction. We speculate that DUF-B may protect the LPMO active site from oxidative damage, a feature that could prove crucial in its ecological and pathogenic contexts.