Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae

Abstract

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 peculiar 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. Notably, EcLPMO displayed a particularly strong synergistic effect with the unimodular chitinase EcChiA, leading to up to 14-fold and 60-fold increases 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. Unexpectedly, using state-of-the-art structural modelling (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.