Biology subjects

Luciano, D.

Publications and source records attributed to Luciano, D..

5 recordsLinked to original sources

bioRxiv2026

Conformation-Dependent Donor Selectivity in the Xanthan Gum Glycosyltransferase GumK Revealed by AI-Based Docking

The interdomain flexibility of GT-B fold glycosyltransferases regulates substrate binding and catalysis, yet the role of local structural variations in donor substrate specificity remains unclear. GumK, a GT70 enzyme from Xanthomonas campestris, exhibits local plasticity within its donor-binding pocket. We classify this plasticity into two conformational states, defined by the presence (closed state) or absence (open state) of a conserved hydrophobic interaction stabilizing the pocket. Using the AI-enhanced docking approach GNINA, we investigated the relationship between these states and substrate specificity by comparing UDP-glucuronate with five acidic and neutral ligand analogs. While docking scores showed limited discrimination among ligands, distance-based analysis between the sugar C6 atom and Lys307 revealed conformation-dependent trends. In the open state, negatively charged sugars preferentially interact with Lys307 via their carboxylate groups. Conversely, the closed state favors interactions with the pyrophosphate moiety. To provide orthogonal validation, we generated ensembles of GumK-substrate complexes using AI-based cofolding methods (AlphaFold3 and Boltz). While Boltz-2 affinities correctly ranked substrate specificity, only the GNINA-generated ensembles were consistent with physics-based simulations in capturing the conformational basis of selectivity. These results suggest that donor specificity arises from the interplay between ligand chemistry and binding-site plasticity rather than from a single rigid binding mode, and highlight complementary strengths of docking and cofolding approaches for studying flexible CAZymes.

biophysics↗

bioRxiv2026

Comparative study of two xanthan gum glycosyltransferases combining AI structure predictions and molecular modeling

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

biophysics↗

bioRxiv2025

Computational characterization of the xanthan gum glycosyltransferase GumK

The activity of GT-B glycosyltransferases depends on their conformational flexibility and high substrate specificity, but the molecular basis of these features is still not well defined. The GT70 family contains a single well-characterized enzyme, GumK, a glucuronosyltransferase from Xanthomonas campestris required for xanthan gum biosynthesis. Here, we applied multiscale molecular simulations and sequence analysis to probe GumK dynamics and substrate specificity. We show that GumK undergoes twisting and bending motions constrained by interdomain contacts and modulated by membrane anchoring. Acceptor-substrate binding within an amphiphilic clamp promotes opening, whereas donor-substrate binding stabilizes closure, defining a substrate-dependent catalytic cycle. Specificity for UDP-glucuronate is mediated by a conserved electrostatic environment centered on Lys307 and a hydrophobic triad that orients the sugar moiety. On the acceptor side, the binding site selectively accommodates polyisoprenyl carriers up to three isoprene units in length and wraps around the substrate, constraining the trisaccharide moiety in a catalytically competent conformation. Comparative analysis highlights GumK-specific motifs that distinguish it from homologous GTs. This work provides mechanistic insight into the GT70 family and the dynamic behavior of GT-B enzymes, establishing principles for the rational engineering of GumK to modify the monosaccharide composition of xanthan gum. Author summaryGlycosyltransferases are enzymes that build many of the sugars and polysaccharides essential for life. One of them, called GumK, is responsible for a key step in producing xanthan gum -- a natural polymer widely used as a thickener in food and industrial materials. Despite its importance, how GumK works at the molecular level has remained unclear. In our study, we used multiscale computer simulation strategies to explore the dynamics of this enzyme and how it interacts with the cell membrane and its sugar substrates. We found that GumK acts like a flexible clamp that opens and closes as it binds two different sugar molecules through specific residues that may control the enzymes selectivity. We also discovered how the surrounding membrane helps the enzyme remain in the correct orientation to perform its function. By identifying the molecular details that determine GumKs selectivity and flexibility, our work provides a foundation for modifying this enzyme to produce xanthan gum with new properties. More broadly, our findings help explain how a large family of related enzymes controls sugar transfer in living organisms, with potential applications in biotechnology and materials science.

biophysics↗

bioRxiv2025

Development of a Vibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes

Polyethylene terephthalate (PET) is a major contributor to plastic waste. Enzymatic PET degradation offers a sustainable recycling approach, but screening for effective enzymes remains challenging. Herein, we established an experimental plate-clearing assay using Vibrio natriegens, exploiting its protein secretion system to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assays robustness, we tested mutants of Fusarium solani pisi cutinase (FsC) and found that one mutant, T45P, exhibited threefold higher activity and a TPA-to-MHET ratio than the wild-type. As a further test case, we screened an error-prone PCR library of FsC using the assay, obtaining a 25% hit rate after screening 150 colonies. This demonstrates the scalability of the method for screening PET-hydrolytic activity in a large number of enzymes.

biochemistry↗

bioRxiv2023

Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia

BackgroundVoltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As mitochondrial gatekeepers, VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. ResultsWe investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). ConclusionsOur in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.

genomics↗