Biology subjects

McKee, L. S.

Publications and source records attributed to McKee, L. S..

4 recordsLinked to original sources

bioRxiv2026

Molecular basis of protein-glycan cross-linking by CpCBM92A revealed by NMR spectroscopy

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

biophysics↗

bioRxiv2026

Characterization of an α-glucan-binding module from Flavobacterium johnsoniae as a founding member of carbohydrate-binding module family XXX

Carbohydrate-binding modules (CBMs) play crucial roles in carbohydrate-active enzymes by promoting substrate recognition and proximity, particularly for insoluble polysaccharides. Here, we report the discovery and characterization of a novel {beta}-trefoil structured CBM associated with a GH87 -1,3-glucanase from Flavobacterium johnsoniae, which accordingly was designated FjCBMXXXGH87. The full-length enzyme efficiently hydrolyzed -1,3-glucan (mutan) and -1,3/-1,6-glucan (alternan), whereas the catalytic domain alone displayed reduced activity, indicating that FjCBMXXXGH87 enhances substrate interaction. Pull-down assays confirmed that FjCBMXXXGH87 binds -1,3-linked glucans, and structural investigation together with site-directed mutagenesis identified two distinct binding sites essential for protein-ligand interactions. Phylogenetic analysis showed that CBMXXX homologs are present together with enzymes from families GH87, GH13, GH16, and GH99, and potentially may comprise up to three binding sites. Together, these findings establish FjCBMXXXGH87 as the founding member of a new CBM family, which may have broad functional versatility in polysaccharide recognition. This discovery expands the repertoire of {beta}-trefoil CBMs and provides new insights into carbohydrate recognition strategies relevant to -glucan degradation.

biochemistry↗

bioRxiv2024

Transcriptomic analysis of Picea abies tissue culture reveals the impact of culture conditions and the presence of glucuronoxylan on extracellular lignin production

Tissue cultures are an important study model for woody plant tissue and can be used to study lignin biosynthesis. The greatest disadvantage of protocols based on extraction of lignin from wood biomass is the almost inevitable alteration of the native structure of lignin. Using a Norway spruce tissue culture with the ability to secrete monolignols into a liquid culture medium, fundamental aspects of lignin have been studied in the past, such as its structure, the enzyme activity related to its polymerization, and its interactions with a secondary cell wall hemicellulose. In this study, parameters that can induce monolignol production and secretion in the tissue culture are investigated via gene expression analysis. The impact of the composition of the solid growth medium, which was in some cases supplemented with xylan, was studied in depth through transcriptomic investigation. We find that the state (i.e. liquid or solid) and the xylan content of the medium can impact gene expression, although microscopic analysis suggests that cellular morphology is consistent. Extracellular lignin was collected from a formulation of liquid medium with the same composition as that used for cellular growth, which was previously presumed to be "non-inducing" of lignin biosynthesis. Chemical analysis of this lignin was performed using nuclear magnetic resonance spectroscopy and size exclusion chromatography, which revealed changes in its structure compared to the polymer produced in the previously developed "inducing" liquid medium. These experiments show that there is still much we do not understand about an oft-used tissue culture system, but show the way to a deeper understanding of the genetic control of lignin biosynthesis.

plant biology↗

bioRxiv2020

Human gut Faecalibacterium prausnitzii deploy a highly efficient conserved system to cross-feed on β-mannan-derived oligosaccharides

{beta}-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of {beta}-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here we show that a dominant butyrate-producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various {beta}-mannooligosaccharides ({beta}-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two {beta}-mannooligosaccharides ({beta}-MOS) utilization loci (FpMULs) supported a concerted model whereby the imported {beta}-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degrader Bacteroides ovatus on polymeric {beta}-mannan resulted in syntrophic growth and production of butyrate, thus confirming the high efficiency of the FpMULs uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of {beta}-mannans metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut. ImportanceCommensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic and detailed biochemical analyses this work reveals the mechanism enabling F. prausnitzii, as a model clostridial cluster IV Firmicute, to cross-feed and access {beta}-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of {beta}-mannans/{beta}-MOS as a common dietary component. Our findings provide a mechanistic understanding of the {beta}-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, thus butyrate production, in the gut.

microbiology↗