Lytic polysaccharide monooxygenases (LPMOs) are multidomain enzymes widely distributed among human pathogenic bacteria. Beyond their polysaccharide-oxidizing activity, multidomain LPMOs are increasingly recognized for roles in host interactions and in facilitating virulence. Notably, immunization of mice with the trimodular LPMO CbpD from Pseudomonas aeruginosa provided protection against lethal P. aeruginosa infection, supporting a link between certain LPMOs and pathogenicity.
In intestinal pathogens, LPMOs are commonly found as tetra- and pentamodular proteins. Their contributions to host interactions have primarily been attributed to carbohydrate recognition, for example binding to N-acetylglucosamine-containing glycans found on host surfaces. However, new structural and biochemical considerations have prompted re-evaluation of additional roles that non-catalytic domains may play in the intestinal environment.
Recent structural predictions and binding studies indicated that the non-catalytic third domain of the Vibrio cholerae colonization factor GbpA harbors a putative copper-binding site. Concurrently, MUC2—the major intestinal mucin—was shown to contain two conserved and distinct copper-binding sites for Cu(II) and Cu(I). Based on these observations, the study hypothesized that auxiliary non-catalytic copper-binding domains in intestinal LPMOs evolved in response to the intestinal mucosal copper landscape, potentially linking these LPMOs to copper handling in the host environment.
To test the hypothesis, the authors examined GbpA and homologous multidomain LPMOs from food-borne intestinal disease–causing Gram-positive bacteria in the genera Bacillus and Listeria. Their approach combined targeted mutagenesis with biochemical assays, spectroscopic characterization, and computational analyses. AlphaFold structural predictions and substrate-binding data informed the identification of potential copper-binding motifs, while experimental alteration of candidate residues allowed assessment of copper interaction and structural consequences. The ensemble of methods was aimed at demonstrating copper-binding capacity and defining structural features of the non-catalytic third domains.
The combined experimental and computational work demonstrated that these multidomain intestinal LPMOs contain conserved copper(I)-binding motifs located on their non-catalytic third domain. While motifs were conserved in the sense of recurring copper-binding capability across different proteins and species, the study found structural distinctions among motifs—i.e., the exact architecture and coordinating residues varied between homologues. The presence of conserved functionality alongside structural diversity suggests evolutionary pressure to maintain copper interaction while allowing lineage-specific variation in domain fold or residue arrangement.
Although the study emphasized conservation of the copper(I)-binding trait, it also documented that the structural realization of the motif is not identical across all examined LPMOs. AlphaFold predictions supported the existence of copper-coordinating pocket(s) in the third domain of GbpA and homologues, and the mutagenesis and spectroscopic analyses confirmed copper(I) interaction in these non-catalytic modules. The findings indicate a recurring design principle—an auxiliary domain specialized for Cu(I) binding—implemented in structurally distinct ways in different bacterial lineages.
Alongside primary experimental results, the authors performed a comprehensive review and reassessment of the literature spanning approximately two decades. This review identified consistent links between the copper-binding ability of multidomain LPMOs and host–pathogen interactions reported previously. Collectively, the literature and the present data align to suggest that copper-binding by LPMO auxiliary domains is a recurring feature associated with colonization and virulence-related processes in intestinal pathogens.
By demonstrating conserved copper(I)-binding motifs in the non-catalytic third domains of intestinal LPMOs, the study connects these proteins to copper management within the intestinal environment. Given that the major intestinal mucin MUC2 binds copper species, the presence of bacterial proteins capable of binding Cu(I) near the mucosal surface provides a plausible mechanistic link between bacterial LPMOs and host copper pools. This connection offers an explanation for why this particular multidomain architecture has been preserved across phylogenetically distinct bacteria that cause intestinal disease: auxiliary copper-binding domains may facilitate survival, colonization, or modulation of local metal availability during infection.
The work presents evidence that multidomain LPMOs from several intestinal pathogens possess conserved yet structurally diverse copper(I)-binding motifs in their non-catalytic third domains. Experimental confirmation through mutagenesis, biochemical, spectroscopic, and computational methods supports a role for these domains in interacting with intestinal copper. A broad literature reassessment reinforces consistent associations between LPMO copper-binding and host–pathogen interactions. Taken together, these observations link LPMO architecture to intestinal copper homeostasis and provide a rationale for the evolutionary conservation of these auxiliary copper-binding domains among intestinal bacterial pathogens.
Note: This article is a preprint and has not undergone peer review. The source reports no competing interests declared by the authors.