Biology subjects

Hausinger, R. P.

Publications and source records attributed to Hausinger, R. P..

5 recordsLinked to original sources

bioRxiv2026

SpyTag-Enabled Assembly of Bacterial Microcompartment Trimers into Macroscopic Layered Protein Materials

Protein self-assembly enables precise nanoscale organization but rarely translates into macroscopic materials that retain functionality beyond aqueous environments. Here, we report that a bacterial microcompartment (BMC) trimer fused with SpyTag (T1-SpyTag), when expressed as a standalone component, undergoes rapid and spontaneous self-assembly into macroscopically visible fibers and layered sheets. These structures span from the nanoscale to the millimeter scale, forming robust three-dimensional protein materials that remain structurally intact and functionally accessible in both solution and dried states. Unlike previously reported SpyTag-enabled BMC systems that function primarily as passive cargo-loading modules, T1-SpyTag macromolecular structures exhibit emergent material behavior, including chemical robustness under denaturing conditions, while preserving covalent reactivity toward SpyCatcher-fused cargos. The multilayered architecture enables tunable surface display, access to ultrathin, processable protein films, and surface renewability through layer-by-layer removal and regeneration. This work demonstrates how a minimal genetic modification of a native protein building block can drive the formation of functional, macroscopic protein materials, thus expanding the design space of BMC-derived assemblies for biointerfaces, catalysis, and sustainable protein materials engineering.

biochemistry↗

bioRxiv2026

Multi-lab, Multi-enzyme Study Demonstrates the Versatility of Bacterial Microcompartment Shells as a Modular Platform for Confined Biocatalysis

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d34f8dorg.highwire.dtl.DTLVardef@10bba9corg.highwire.dtl.DTLVardef@10868b7org.highwire.dtl.DTLVardef@112cb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Structural Basis for Catalysis and Substrate Specificity of a LarA Racemase with a Broad Substrate Spectrum

The LarA family consists of diverse racemases/epimerases that interconvert the diastereomers of a variety of -hydroxyacids by using a nickel-pincer nucleotide (NPN) cofactor. The hidden redox reaction catalyzed by the NPN cofactor makes LarA enzymes attractive engineering targets for applications. However, how a LarA enzyme binds its natural substrate and recognizes different -hydroxyacids has not been elucidated. Here, we report three high-resolution structures of the enzyme-substrate complexes of a broad-spectrum LarA enzyme from Isosphaera pallida (LarAIp). The substrate binding mode reveals an optimal orientation and distance between the hydride donor and acceptor, strongly supporting the proposed proton-coupled hydride transfer mechanism. The experimentally solved structures, together with the structural models of other LarA enzymes, allow us to identify the residues/structural elements critically involved in the interactions with different -hydroxyacid substrates. Collectively, this work provides a critical structural basis for catalysis and substrate recognition of the diverse enzymes in the LarA family, thus building a foundation for enzyme engineering.

biochemistry↗

bioRxiv2022

Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor

The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarALp) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarALp, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH4) irreversibly inactivates LarALp accompanied by a rapid color change of the enzyme. The drastically altered ultraviolet-visible spectra during NaBH4 titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the mechanism-based inactivation. High resolution structures of LarALp revealed a substantially weakened C-Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarALp inactivation by NaBH4 that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.

biochemistry↗

bioRxiv2020

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and function

The presence of lanthanide elements (Ln3+) and pyrroloquinoline quinone (PQQ) containing cofactors in XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs) has expanded the list of biological elements and opened novel areas of metabolism and ecology. Other MDHs known as MxaFIs are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what protein features distinguish enzymes using Ln3+-PQQ cofactors from those that do not. In this study, we investigated the functional importance of a proposed lanthanide-coordinating aspartate using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1. We report two crystal structures of XoxF1, one containing PQQ and the other free of this organic molecule, both with La3+ bound in the active site region and coordinated by Asp320. Using constructs to produce either recombinant XoxF1 or its D320A variant, we show Asp320 is needed for in vivo catalytic function, in vitro activity of purified enzyme, and coordination of La3+. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinate Ca2+, but exhibit little or no catalytic activity. In addition, we generated the parallel substitution to produce ExaF D319S, and showed the enzyme loses the capacity for efficient ethanol oxidation with La3+. These results provide empirical evidence of an essential Ln3+-coordinating aspartate for the function of XoxF MDHs and ExaF EDHs; thus, supporting the suggestion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

biochemistry↗