Biology subjects

Rabe, P.

Publications and source records attributed to Rabe, P..

6 recordsLinked to original sources

bioRxiv2026

Biochemical characterisation of fungal bioluminescence enzymes reveals substrate inhibition and secondary turnover of 3-hydroxyhispidin by H3H

The fungal bioluminescence pathway enables autonomous light production from caffeic acid and has emerged as a versatile platform for bioimaging and synthetic biology. However, biochemical characterisation of its core enzymes remains limited. Here, we establish recombinant purification and quantitative in vitro characterisation of fungal luciferase (Luz) and hispidin-3-hydroxylase (H3H) homologues from Neonothopanus nambi and Mycena chlorophos. Complementary luminescence and mass spectrometry-based assays revealed pronounced substrate inhibition of H3H by hispidin, conserved across both homologues. Direct monitoring of substrate turnover further revealed an unexpected secondary H3H-catalysed conversion of 3-hydroxyhispidin that remained dependent on NADPH and FAD. NMR, isotope labelling and product characterisation support an additional oxidative transformation followed by formation of multiple downstream products, including caffeic acid, thereby reconnecting secondary turnover with an upstream intermediate of the pathway. H3H nevertheless retained a strong kinetic preference for hispidin over 3-hydroxyhispidin. Together, these findings reveal previously unrecognised catalytic complexity within the fungal bioluminescence pathway and provide a biochemical framework for understanding and engineering pathway flux.

biochemistry↗

bioRxiv2026

Design and Evolution of an Orthogonal HaloTag for Multiplexed Labeling in Cells

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

biochemistry↗

bioRxiv2025

Drop on fixed target reaction initiation approach for serial and time resolved crystallography

We describe the design and implementation of a drop on fixed target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume (40 - 90 pL) aqueous droplets, containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals immobilised on a solid support. The system was tested with various enzyme systems, including lysozyme and two -lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free electron laser facilities was evaluated. This drop on fixed target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

biophysics↗

bioRxiv2025

Targeting Mycobacterial Transpeptidases: Evaluating the Roles of Ldt and PBP Inhibition in Suppressing Mycobacterium smegmatis

{beta}-Lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimisation of the classical {beta}-lactam scaffolds or development of new or non-{beta}-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of {beta}-lactams include L,D-transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the {beta}-lactam antibacterial mechanisms are not understood. We used fluorogenic substrate mimics to investigate the effects of {beta}-lactams and reported LdtMt2 inhibitors on Mycobacterium smegmatis (Msm), assessing their impacts on the transpeptidase activities of Ldts and PBPs in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; though under the tested conditions a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, inhibition of both PBPs and Ldts was observed in all active inhibitors, though {beta}-lactams manifest increased potency of PBP inhibition. The combination of the {beta}-lactams meropenem and faropenem with selected LdtMt2 inhibitors manifested an additive inhibitory effect against Msm. Our results highlight the importance of further optimising PBPs and Ldt transpeptidase inhibition, particularly of Ldts, to improve {beta}-lactam efficacy versus mycobacteria.

biochemistry↗

bioRxiv2024

Cardiac stress leads to regulation of Filamin C dimerisation via an ancient phosphorylation-modulated interaction with HSPB7

The biomechanical properties and responses of tissues underpin a variety of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. Seemingly a multi-functional protein that interacts with a variety of partners, how FLNC is regulated at the molecular level is not well understood. Here we have investigated its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We found that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we have solved by means of X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work rationalises on the molecular level how FLNC might switch between stabilising functions in the cell, and reveals how HSPB7 acts as a specific molecular chaperone that regulates FLNC.

molecular biology↗

bioRxiv2021

The termite fungal cultivar Termitomyces combines diverse enzymes and oxidative reactions for plant biomass conversion

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using pre-digested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive OMICs and activity-based evidence that Termitomyces partially depolymerizes lignocellulose through the combined actions of high-redox potential oxidizing enzymes (laccases, aryl-alcohol oxidases and a manganese peroxidase), the production of extracellular H2O2 and Fenton-based oxidative degradation, which is catalyzed by a newly described 2-methoxybenzoquinone/hydroquinone redox shuttle system and mediated by secreted chelating dicarboxylic acids. In combination, our approaches reveal a comprehensive depiction of how the efficient biomass degradation mechanism in this ancient insect agricultural symbiosis is accomplished through a combination of white- and brown-rot mechanisms. ImportanceFungus-growing termites have perfected the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a co-diversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here we provide genomic, transcriptomic and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton-based hydroquinone-catalyzed lignin-degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms open new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

microbiology↗