Biology subjects

Vermaas, J. V.

Publications and source records attributed to Vermaas, J. V..

14 recordsLinked to original sources

bioRxiv2026

Small but Mighty: The Surprising Metabolic Range of a Bacterial Microcompartment

Bacterial microcompartments (BMCs) are protein-based metabolic modules increasingly investigated as potential intracellular nanoreactors. In this study, we engineered Escherichia coli by chromosomally integrating the propanediol utilization (pdu) operon from Citrobacter freundii and evaluated the metabolic versatility of the heterologously produced Pdu BMCs. The formation of well-assembled, operational Pdu BMCs was confirmed in cellula. Oxygen was found to inhibit 1,2-propanediol utilization, an effect alleviated under microaerobic conditions. The engineered E. coli strain assimilated a range of diols and triols, demonstrating that Pdu BMCs process both their native substrate and glycerol and linear diols up to 1,2-butanediol (C4). This substrate promiscuity was exploited to produce high value-added compounds and revealed previously unrecognized potential for plastic upcycling through ethylene glycol metabolism. In keeping with molecular dynamics simulations indicating that the Pdu BMC shell does not impose significant diffusion barriers for longer chain diols and triols, the range of amenable substrates was successfully extended to 1,2-pentanediol (C5) by mutating the diol dehydratase large subunit (PduC). Together, these findings establish heterologous Pdu BMCs as promiscuous and genetically tunable metabolic modules in E. coli, highlighting their potential as scalable platforms for synthetic biology and biotechnological applications. ImportanceBacterial microcompartments (BMCs) are increasingly popular modular platforms for organizing and optimizing metabolic pathways, yet their substrate scope and engineering potential remain underexplored. Here, we demonstrate that heterologous Pdu BMCs expressed in Escherichia coli metabolize a substantially broader range of substrates than previously appreciated, including various diols and triols outside their native range. We further show that BMC performance is modulated by oxygen availability and can be predictably expanded by targeted enzyme engineering. These findings establish Pdu BMCs as flexible, genetically tunable metabolic modules capable of converting diverse feedstocks into value-added products, while also revealing an unanticipated capacity for plastic upcycling via ethylene glycol utilization. By integrating pathway engineering, physiological characterization, and computational modelling, this work advances the design of BMC-based systems for sustainable bioproduction and highlights their potential as customizable intracellular nanoreactors for biotechnology.

biochemistry↗

bioRxiv2026

Molecular design principles for Photosystem I-based biohybrid solar fuel catalysts

Direct solar-to-chemical conversion offers a compelling route to clean, dispatchable energy. Photosystem I (PSI), an evolutionarily optimized light-driven oxidoreductase central to oxygenic photosynthesis, can be repurposed for direct solar-fuel production by efficiently coupling its photochemistry to catalysts, thereby storing sunlight as chemical energy in the H-H bond of H2. One promising architecture integrates PSI with Pt nanoparticle (PtNP) catalysts to create photocatalytic PSI-PtNP biohybrids. Advancing these systems requires molecular-level insight into protein-nanoparticle interactions and the bio-nano electron transfer pathways that govern activity; however, progress has been constrained by limited structural data to guide rational design. Here, we present two molecular structures of active PSI-PtNP assemblies that (a) compare thermophilic and mesophilic PSI scaffolds and (b) probe how removal of the terminal [4Fe-4S] clusters and stromal subunits in PSI reshapes protein-nanoparticle interfaces and photocatalysis. Structural analyses and molecular dynamics simulations define the interface topology, electrostatics, and cofactor-to-nanoparticle distances, revealing key molecular features that control biohybrid formation and electron transfer efficiency. These data establish mechanistic links between scaffold composition, bio-nano interface geometry, and catalytic performance, yielding design principles for optimizing PSI-PtNP architectures. The resulting structure-function insights provide a blueprint for engineering PSI-based solar-fuels systems and, more broadly, inform the design of protein-nanomaterial interfaces for light-driven catalysis.

biophysics↗

bioRxiv2026

Encapsulation in a bacterial microcompartment shell improves thermal stability of a glycolytic enzyme

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMCs) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62{degrees}C.

synthetic biology↗

bioRxiv2025

Oxidative Stress-Responsive Cell Wall Remodeling Depends on Phosphate in Candida albicans

The growing number of patients susceptible to invasive Candida albicans infections has intensified the need for new antifungal targets in pathways essential for fungal growth and pathogenesis. Among these pathways, phosphate homeostasis has emerged as a significant determinant of virulence, yet how phosphate availability shapes cell wall structure in response to host-derived oxidative stress remains unclear. During commensal growth, C. albicans cells typically enjoy phosphate repletion and a neutral redox environment. Transitioning to invade host tissues, they simultaneously experience phosphate deprivation and intense extrinsic oxidative stress. Here, we employ solid-state NMR to render details of cell wall remodeling in response to oxidative stress, in its dependence on phosphate. Phosphate deprived cells remodel the rigid wall core and reduce hydration and polymer mobility in the absence of oxidative stress. During hydrogen peroxide exposure, highly mobile outer polysaccharides are primary interactors. In wildtype cells, some of these polymers are recruited into the rigid core, reinforcing the wall scaffold, whereas phosphate transport mutants fail to undergo this remodeling. These findings establish phosphate acquisition as a component of oxidative defense and link nutrient sensing and -availability to the mechanical resilience of the fungal cell wall, revealing an architectural vulnerability with relevance for antifungal development.

microbiology↗

bioRxiv2025

Screening for residues in Atg11, a central organizer of selective autophagy in yeast, important for binding with Atg9

The yeast protein Atg11, whose structure is unknown, is a central organizer of autophagosome formation that recruits Atg9 during selective autophagy. Although the residues in Atg9 responsible for this interaction are known, those in Atg11 are not. In an attempt to discover the binding site of Atg9 on Atg11, we screened a number of mutants within amino acid residues 455-627 of Atg11, guided in part by an AlphaFold2-generated model of the Atg11 dimer. However, we were not able to identify specific residues essential for the interaction with Atg9, suggesting that the binding region may lie elsewhere on Atg11.

biochemistry↗

bioRxiv2025

IRE1 Regulates TOR Signaling via RIDD of RAPTOR1b to Coordinate Growth and Stress Adaptation

Plant growth and stress resilience depend on integrating diverse signals into coordinated cellular responses. The endoplasmic reticulum (ER) stress sensor IRE1 maintains ER homeostasis and modulates Target of Rapamycin (TOR) signaling. Here, we reveal that TOR misregulation in an ire1ab mutant reduces sensitivity to the stress hormone abscisic acid (ABA), mediated by TOR-dependent phosphorylation of the ABA receptor PYL1. Further, we show that IRE1s endonuclease activity is required for TOR regulation, acting independently of the canonical IRE1/bZIP60 unfolded protein response. Instead, it occurs via Regulated IRE1- Dependent Decay (RIDD) of specific transcripts. We identify RAPTOR1b as a direct RIDD target, establishing a mechanistic link between ER stress sensing and TOR signaling. RIDD- mediated degradation of RAPTOR1b mRNA is required for appropriate ABA responses and stress adaptation. These findings uncover a noncanonical IRE1-TOR signaling axis that fine- tunes growth and stress responses through selective mRNA decay.

plant biology↗

bioRxiv2024

Structure Characterization of Bacterial Microcompartment Shells via X-ray Scattering and Coordinate Modeling: Evidence for adventitious capture of cytoplasmic proteins

Bacterial microcompartments (BMCs) are self-assembling, protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small angle X-ray scattering (SAXS) and pair density distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum that were isolated following expression in E. coli. The analysis allowed BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli. Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

biophysics↗

bioRxiv2024

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

microbiology↗

bioRxiv2024

Advancing thermostability of the key photorespiratory enzyme glycerate 3-kinase by structure-based recombination

As global temperatures rise, maintaining and improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalyzing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. Exploring inter-specific variation of the key photorespiratory enzyme glycerate kinase (GLYK) from various photosynthetic organisms, we found that the homolog from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand factors influencing thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, a series of hybrid proteins were produced and analyzed. These hybrid enzymes contained selected loop regions from CmGLYK replacing the most highly mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 {degrees}C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. While this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing thermostability of other plant enzymes.

biochemistry↗

bioRxiv2024

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria which encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semi-permeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We use molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

biophysics↗

bioRxiv2023

Structural Remodeling of Fungal Cell Wall Promotes Resistance to Echinocandins

The insufficient efficacy of existing antifungal drugs and the rise in resistance necessitate the development of new therapeutic agents with novel functional mechanisms1,2. Echinocandins are an important class of antifungals that inhibit {beta}-1,3-glucan biosynthesis to interfere with cell wall structure and function3,4. However, their efficacy is limited by the fungistatic activity against Aspergillus species and the trailing effect during clinical application. Here, we describe how echinocandins remodel the supramolecular assembly of carbohydrate polymers in the fungal cell wall in an unexpected manner, possibly resulting in a subsequent inhibition of the activity of these drugs. Solid-state nuclear magnetic resonance (ssNMR) analysis of intact cells from the human pathogenic fungus Aspergillus fumigatus showed that the loss of {beta}-1,3-glucan and the increase of chitin content led to a decrease in cell wall mobility and water-permeability, thus enhancing resistance to environmental stresses. Chitosan and -1,3-glucan were found to be important buffering molecules whose physical association with chitin maintained the wall integrity. These new findings revealed the difficult-to-understand structural principles governing fungal pathogens response to echinocandins and opened new avenues for designing novel antifungal agents with improved efficacy.

biophysics↗

bioRxiv2020

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N{delta} (HD) and N{epsilon} (HE) protonation of His41 and His164, respectively, the -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

biophysics↗

bioRxiv2020

Data-guided Multi-Map variables for ensemble refinement of molecular movies

Driving molecular dynamics simulations with data-guided collective variables offer a promising strategy to recover thermodynamic information from structure-centric experiments. Here, the 3-dimensional electron density of a protein, as it would be determined by cryo-EM or X-ray crystallography, is used to achieve simultaneously free-energy costs of conformational transitions and refined atomic structures. Unlike previous density-driven molecular dynamics methodologies that determine only the best map-model fits, our work uses the recently developed Multi-Map methodology to monitor concerted movements within equilibrium, non-equilibrium, and enhanced sampling simulations. Construction of all-atom ensembles along chosen values of the Multi-Map variable enables simultaneous estimation of average properties, as well as real-space refinement of the structures contributing to such averages. Using three proteins of increasing size, we demonstrate that biased simulation along reaction coordinates derived from electron densities can serve to induce conformational transitions between known intermediates. The simulated pathways appear reversible, with minimal hysteresis and require only low-resolution density information to guide the transition. The induced transitions also produce estimates for free energy differences that can be directly compared to experimental observables and population distributions. The refined model quality is superior compared to those found in the Protein DataBank. We find that the best quantitative agreement with experimental free-energy differences is obtained using medium resolution (~5 [A]) density information coupled to comparatively large structural transitions. Practical considerations for generating transitions with multiple intermediate atomic density distributions are also discussed.

biophysics↗

bioRxiv2018

Characterizing Activity and Thermostability of GH5 Cellulase Chimeras from Mesophilic and Thermophilic Parents

Cellulases from glycoside hydrolase (GH) family 5 are key enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical ({beta})8-barrel structure, where each ({beta}) module is essential for the enzyme stability and activity. Despite their shared topology, the thermostability of GH5 enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on a previously characterized thermophilic GH5 cellulase from Talaromyces emersonii (TeEgl5A, with an optimal temperature of 90{degrees}C), we created ten hybrid enzymes with the mesophilic cellulase from Prosthecium opalus (PoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optima (10 and 20{degrees}C), T50 (15 and 19{degrees}C), Tm (16.5 and 22.9{degrees}C), and extended half life, t1/2 (~240- and 650-fold at 55{degrees}C) relative to the mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 cellulase from Aspergillus nidulans (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics simulations (MD) of both PoCel5 and TeEgl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic packing of the interface between 2 and 3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to the mesophilic parent PoCel5.\n\nIMPORTANCEThermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM-barrel fragments and even fragments from unrelated folds, to generate new structures. However, there are not many research on GH5 cellulases. In this study, two GH5 cellulases, which showed TIM-barrel structure, PoCel5 and TeEgl5A with different thermal properties were hybridized to study the roles of different ({beta}) motifs. This work illustrates the role that structure guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

bioengineering↗