ACS Catalysis

Glycoside hydrolases (GHs) play central roles in carbohydrate metabolism and are widely exploited for industrial and biomedical applications. However, they are often not optimal for applications due to their constrained function and strict stereochemical specificity, necessitating the discovery and optimization of distinct enzymes for each glycosidic configuration. Members of glycoside hydrolase family 1 (GH1) are archetypal retaining {beta}-glycosidases, while -specific activity is rare within this family. Here, I demonstrate that a retaining GH1 enzyme can be engineered to hydrolyze both {beta}- and -configured substrates without altering its canonical catalytic residues. Using a well-characterized {beta}-glycosidase and computational protein design strategies targeting second-shell residues surrounding the active site, a bifunctional {beta}-/-glycosidase containing 45 mutations was generated. The engineered variant acquired the ability to hydrolyze the -configured substrate 4-nitrophenyl--D-glucopyranoside while retaining activity toward the originals {beta}-substrates, with reduced catalytic efficiency and thermostability. Structural modeling and docking analyses reveal that the engineered enzyme preserves the original fold and accommodates substrates within the catalytic pocket in a similar manner to the wild type. These findings provide direct evidence that stereochemical constraint in retaining GH is more flexible than previously appreciated and can be modulated through targeted engineering.

Enzyme-directed evolution increasingly relies on computational tools to prioritize mutations, yet their practical value is difficult to assess because kinetic data are often aggregated across heterogeneous assay conditions, inflating apparent generalization. Here we introduce EnzyArena, a curated benchmark that groups kinetic parameters (kcat, Km, kcat/Km) into condition-matched experimental subsets to enable realistic evaluation. Using this resource, we benchmark 10 representative models from two arising strategy families--zero-shot fitness prediction and supervised kinetic-parameter prediction--across BRENDA- and SABIO-RK-derived subsets and 25 independent mutagenesis datasets. Kinetic-parameter predictors perform strongly on database-derived subsets but lose their advantage on independent datasets, whereas zero-shot predictors show more consistent generalization. A simple consensus of multiple zero-shot models further improves the precision of identifying beneficial mutants. We prospectively validated these findings in a wet-lab campaign (150 mutants) comparing random mutants, UniKP-prioritized mutants and ESM-1v-prioritized mutants (representing supervised kinetic-parameter prediction and zero-shot fitness prediction, respectively), where ESM-1v achieved the highest utility and UniKP underperformed the random baseline. Together, this study establishes realistic baselines for computational mutant prioritization and highlights consensus zero-shot strategies as a practical starting point for enzyme engineering.

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

Methyl-coenzyme M reductase (MCR) is a crucial enzyme for methanogenesis and harbors several unusual post-translational modifications. Recent studies have identified glutamine C-methyltransferase (QCMT), as a B12-dependent radical SAM enzyme responsible for methylating a glutamine residue within the MCR active site. B12-dependent radical SAM enzymes have the remarkable ability to alkylate unactivated Csp2- and Csp3-atoms in a stereoselective manner. However, the factors influencing the stereo-selectivity and catalytic properties of this emerging superfamily of enzymes remain poorly understood. In this study, we report the mechanistic, structural, and biochemical investigation of several QCMTs. Our findings reveal significant differences among them, notably in their ability to bind cobalamin. In addition, our data support that C H-atom abstraction and methyl transfer are not concerted but rather independent processes that require motion within the enzymes active site. We also demonstrate that QCMT can catalyze novel reactions, including the formation of unnatural C-methylated residues, peptide epimerization, reversible H-atom abstraction, and the direct conversion of glycine into O_SCPLOWDC_SCPLOW-alanine. Overall, our data are consistent with QCMT being a unique and versatile biocatalyst allowing for the installation of unnatural post-translational modifications and provide a structural and biochemical rationale for the control of the stereochemistry by B12-dependent radical SAM enzymes.

Macrolides are an antibiotic class widely used in both human and veterinary medicine, and function by interfering with protein synthesis. Regrettably, numerous strategies for evading the antibiotic properties of macrolides have been found in bacteria, including enzyme-mediated inactivation. These mechanisms are now widely disseminated among pathogenic, animal-associated and environmental bacteria making them a One Health issue. Macrolide esterases, which hydrolyze the macrolactones ester bond, confer one such resistance mechanism. Two types of macrolide esterases have thus far been identified, the well-studied erythromycin esterases and the recently discovered Est-type enzymes that belong to the /{beta}-hydrolase superfamily. We present detailed structure-function studies for four diverse Est type esterases: which only share 44-66% sequence identity (EstTSf, EstTSt, EstTBc, and EstXEc). In addition to resistance profiling and substrate specificity studies, we present structures for all four enzymes, including structures for EstTBc and EstXEc in complex with tylosin and tylvalosin macrolides, post hydrolysis. Complementing the data with mutational and kinetic studies allowed for a detailed analysis of the structural basis for macrolide-enzyme interactions. Combined the data suggest that promiscuous binding and imprecise positioning, mediated by a water-cage, dictate substrate specificity for Est-type macrolide resistance enzymes. These insights may prove beneficial for next-generation antibiotic development.

Metabolic engineering to produce molecules not naturally synthesized by the host often requires directed evolution to improve pathway enzyme performance. Growth-coupled selection can dramatically increase directed-evolution throughput, and manipulation of redox balance has proven effective for tying reductase fitness to microbial growth. However, most redox-balance selections require feeding the reductase substrate because of stoichiometric constraints. This is impractical for many biosynthetic pathways either due to practical limitations on cost or complexity of bulk substrate synthesis, or the lack of an ability to transport substrate into cells, for example intracellular acyl-CoA/ACP intermediates. Here we define stoichiometric constraints that make substrate feeding necessary for many acetyl-CoA-derived reduction pathways in NADPH-imbalanced hosts. We overcome these constraints with a dual-feedstock strategy in which glucose provides reducing power while acetate supplies additional acetyl-CoA without directly perturbing redox balance. In an engineered E. coli selection strain, acetate co-feeding enabled growth coupling of acetaldehyde, 3-hydroxybutyrate, and mevalonate production and produced a linear correlation between product formation and growth. We then used this selection to evolve a class II HMG-CoA reductase (HMGR) from Delftia acidovorans toward NADPH utilization, enriching variants with improved NADPH-dependent activity. Finally, propionate co-feeding enabled growth coupling of propionyl-CoA reduction, supporting the generality of carbon co-feeding for selecting enzymes in pathways involving acyl-chain elongation and reduction. HighlightsO_LIStoichiometric limits of redox-balance growth coupling are defined C_LIO_LIAcetate co-feeding supplies acetyl-CoA without perturbing redox balance C_LIO_LICo-feeding enables growth coupling of acetaldehyde, 3-HB, and mevalonate C_LIO_LIGrowth coupling enables evolution of HMGR toward NADPH specificity C_LIO_LIPropionate co-feeding extends growth coupling to additional acyl-CoA substrates C_LI

The inositol monophosphatase (IMPase) orthologue is pivotal for virulence, pathogenesis, and biofilm regulation, and is therefore considered a potential drug target in Pseudomonas aeruginosa and other bacterial pathogens. The mammalian IMPase orthologue is an established drug target for bipolar disorder. The precise catalytic mechanism in this class of enzymes remains obscure despite five to six decades of extensive efforts and detailed studies of substrate, transition-state analogue, and product-bound structures. Here, we have solved the crystal structures of the IMPase orthologue from Pseudomonas aeruginosa (PaIMPase), capturing pre- and post-catalytic snapshots of metal-substrate- and metal-product-mimic-bound states. Moreover, we solved the metal-substrate transition-state-analogue-bound crystal structure of the enzyme. Critical evaluation of these high-resolution crystal structures of PaIMPase complexed with substrate, transition-state analogue, and product mimic (myo-inositol and phosphate) supports three Mg2+-dependent catalytic mechanisms of PaIMPase. The structural snapshots indicate that, at the enzyme active site, a metal (Mg2+)-coordinating water molecule, activated by two bound Mg2+ ions and the active-site-proximal Threonine/Aspartate dyad, attacks the central phosphorus atom of the bound substrate, leading to formation of a trigonal bipyramidal transition state. Following that, the immediate breakdown of the P-O bond results in the formation of inositolate and phosphate ions. The second water molecule, activated by another Mg2+ dyad, facilitates the departure of myo-inositol and phosphate from the active site. The detailed mechanistic insights gained from this work may offer a foundation for the rational design of precise inhibitors against PaIMPase.

Polyethylene terephthalate (PET) hydrolysis by Ideonella sakaiensis PETase (IsPETase) begins at a heterogeneous solid-liquid interface, yet the molecular basis of productive surface recognition remains poorly resolved. Here, we combined a Martini 3 coarse-grained PET model with G[o]Martini protein dynamics to investigate IsPETase binding to an extended PET surface. A four-state kinetic model, comprising unbound, encounter, docked, and pre-catalytic states, shows that productive binding is not limited by adsorption itself, but by a post-adsorption re-registration step that converts surface-bound encounter complexes into productively aligned configurations. The simulations reveal a stage-dependent role of conformational flexibility: flexible surface loops facilitate early capture, whereas excessive flexibility promotes misregistered hydrophobic contacts, over-stabilizes non-productive encounter states, and lowers the overall probability of productive commitment. Analysis of productive trajectories further identifies three microscopic reorientation modes by which the enzyme reaches the pre-catalytic state after adsorption. Comparative simulations of engineered PETase variants uncover a flexibility-driven speed-yield trade-off, in which increased flexibility accelerates successful binding events but reduces productive yield through encounter-state over-anchoring. Guided by this mechanism, we formulated a landscape-based design strategy that either weakens encounter-specific anchors or reinforces product-like contacts, leading to mutations that improve productive-binding yield. These results identify post-adsorption alignment as the key kinetic bottleneck in PETase surface recognition and provide a mechanistic framework for designing enzymes that operate at heterogeneous polymer interfaces.

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, especially from extreme environments, and constitute an enormous source of functional proteins. Conservative mining of these data by close sequence homology alone tends to identify merely different versions of known enzymes. Here we present a discovery strategy of meganucleases based on wider capture of less homologous enzymes with new function in metagenomic databases, incorporating metadata with homology, relying on cell-free expression to bypass host incompatibility and the need for purification, along with using deep sequencing for experimental assessment of substrate specificity and cleavage pattern, circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55{degrees}C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate unique 4 base pair palindromic 3'-overhangs -- the first monomeric meganuclease to produce such overhangs. Co-folding models of I-MG11 bound to DNA provide a structural context for enzyme-DNA interactions, highlighting differences from other monomeric LAGLIDADG meganucleases (e.g. I-SceI) shaped by InDels (insertion-deletions) in the DNA binding region that may cause specificity changes. Our strategy streamlines bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the molecular biology toolbox. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=31 SRC="FIGDIR/small/712669v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@885de7org.highwire.dtl.DTLVardef@cce9fdorg.highwire.dtl.DTLVardef@116055borg.highwire.dtl.DTLVardef@b9b59e_HPS_FORMAT_FIGEXP M_FIG C_FIG

CO dehydrogenases (CODH) are metalloenzymes that reversibly oxidize CO to CO2, at a buried NiFe4S4 active site. The substrates, CO and CO2, need therefore to be transported through the protein matrix to reach the active site. The most likely pathway for intra-protein diffusion is the hydrophobic channel identified in the crystal structures. Here, we use site-directed mutagenesis to study the highly conserved isoleucine 563 of Thermococcus sp. AM4 CODH2. Mutations at this position change the biochemical properties (KM for CO, product inhibition constant, catalytic bias...), and increase the resistance of the enzyme to the inhibitor O2, showing that isoleucine 563 indeed lines the gas channel. The I563F mutation decreases the bimolecular rate constant of inhibition by O2 15-fold, and increases the IC50 20-fold, which is the strongest improvement in O2 resistance reported so far. We show that the size of the introduced amino acids is less important than their flexibility - along with the size of the cavity formed near the active site in the channel. We also conclude that O2 access to the active site cannot be slowed down without also affecting CO diffusion. This tradeoff will have to be considered in further attempts to use site-directed mutagenesis to make CODHs more O2 tolerant.

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine-or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions.1-3 All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine.4,5 This knowledge has guided predictions and study of BSH activity in the gut microbiome6,7 as well as the development of BSH inhibitors8. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

Plant-type (Form I) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) suffers from inherent catalytic trade-offs and a strong dependency on other proteins--including an essential small subunit (SSU) and auxiliary chaperones--for assembly, constraining the enzymes evolutionary and engineering potential. Here, we investigated representatives from the newly discovered clade Form IF. These enzymes do not require specific chaperones to form functional complexes, exhibit high CO2-specificities (SC/O [~]50) while maintaining high turnover rates (up to kcat [~]11 s-1). Remarkably, two Form IF representatives (IF-1/IF-2) lost the dependency on the SSU and assemble into homo-octameric complexes without their cognate SSUs. While the SSU is not necessary for catalysis, its addition improves both activity and specificity in IF-1/IF-2. Our results show that complexity is actually not required to achieve highly active, specific and functional Rubisco variants--and that this complexity can even be reverted--which challenges our current thinking on the evolution and catalytic mechanism of Rubisco.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

Combinatorial mutagenesis is essential for exploring protein sequence-function landscapes in engineering applications. However, while large-scale machine learning benchmarks exist for protein function prediction, they are primarily limited to single-mutant libraries, leaving a critical gap for combinatorial mutagenesis. Here we introduce CombinGym, a benchmarking platform featuring 14 curated combinatorial mutagenesis datasets spanning 9 proteins with diverse functional properties including binding affinity, fluorescence, and enzymatic activities. We evaluated nine machine learning algorithms from five methodological categories (alignment-based, protein language, structure-based, sequence-label, and substitution-based) across multiple prediction tasks, assessing both zero-shot and supervised learning performance using Spearmans {rho} and Normalized Discounted Cumulative Gain metrics. Our analysis reveals the substantial impact of measurement noise and data processing strategies on model performance. By implementing hierarchical dataset splits (0-vs-rest, 1-vs-rest, 2-vs-rest, and 3-vs-rest scenarios), we demonstrate the value of lower-order mutation data for empowering machine learning models to predict higher-order mutant properties. We validated this capacity through both in silico simulation (improving fluorescence brightness of an oxygen-independent fluorescent protein) and experimental validation (engineering enzyme substrate specificity), achieving a substantial increase in specific activity. All datasets, benchmarks, and metrics are available through an interactive website (https://www.combingym.org), facilitating collaborative dataset expansion and model development through integration with automated biofoundry platforms.

D-alanylation of teichoic acids is a widespread modification of Gram-positive bacterial cell envelopes that modulates resistance to environmental stresses and host interactions. Although the cytosolic steps of this pathway are well characterized, the extracellular reactions responsible for transferring D-alanine onto teichoic acids remain poorly understood. Here we investigate the role of DltD in the commensal bacterium Lactiplantibacillus plantarum. We determined the 2.3 [A] crystal structure of the extracellular catalytic domain of DltD, which adopts an SGNH-hydrolase fold with a conserved Ser-His-Asp catalytic triad. Docking analyses with lipoteichoic acids (LTA) fragments suggest that the glycerol-phosphate backbone of LTA is accommodated along a surface groove leading to the catalytic serine, with conserved residues contributing to substrate positioning. Biochemical measurements further reveal direct interactions between DltD, the acyl-carrier protein DltX, and the LTA esterase DltE. The conserved C-terminal motif of DltX binds DltD and is required for efficient D-alanylation and for L. plantarum-mediated promotion of Drosophila juvenile growth. Together, these findings support a DltX-dependent acyl-transfer mechanism and reveal an interaction network that coordinates LTA D-alanylation in a symbiotic bacterium.

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@1e56ffborg.highwire.dtl.DTLVardef@1ac8b5org.highwire.dtl.DTLVardef@6f23c1org.highwire.dtl.DTLVardef@945c54_HPS_FORMAT_FIGEXP M_FIG C_FIG

Disulfide-rich miniproteins constitute compact and highly stable scaffolds of growing interest for molecular and structural engineering. Schistosomins are [~]80-residue proteins conserved across gastropods that form a long-standing orphan family whose structure and biological roles have remained unknown. Here, we report the total chemical synthesis and structural characterization of a schistosomin isoform from Biomphalaria glabrata, a medically relevant intermediate host of the parasite Schistosoma mansoni. Using state-of-the-art solid-phase peptide synthesis, chemoselective peptide ligation, and controlled oxidative folding, we obtained homogeneous well folded schistosomin suitable for biophysical and structural studies. High-resolution X-ray crystallography reveals a previously undescribed disulfide-rich fold defining a new class of miniprotein scaffold. Nano differential scanning fluorimetry and circular dichroism experiments demonstrate the remarkable thermal stability of this scaffold, while molecular dynamics simulations confirm the intrinsic rigidity of its disulfide-stabilized core and show that the two naturally occurring isoforms differing by a single residue exhibit nearly indistinguishable structural and dynamic properties. Finally, transcript and protein analyses across snail tissues provide the first spatial expression map of schistosomin in a medically relevant mollusk. Together, this work establishes schistosomin as a novel and robust miniprotein scaffold and provides a structural and biological framework for exploring its function and potential applications.

How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes. ImportanceThis work seeks to understand the basis for oligomerization of a large oxidoreductase that is important for metabolizing sulfur, an essential chemical for all of biology. A 52-residue long leader peptide is necessary and sufficient for assembly into a particularly stable octamer that is resistant to chemical denaturation under diverse conditions.

Antimicrobial resistance is one of the most serious challenges to global health, yet the development of new molecules with novel mechanisms of action to combat resistance is lacking. Here, we report the discovery of molecular glue-like compounds that recruit TEM-family {beta}-lactamases to the bacterial protease DegP for degradation. {beta}-lactamase inhibitor tazobactam was found to accelerate degradation of TEM {beta}-lactamases by DegP, which was further enhanced by linkerless incorporation of dipeptide motifs enriched among DegP substrates. The resulting molecular glue-like degraders showed improved synergy with {beta}-lactam piperacillin against resistant E. coli compared to tazobactam, as well as good pharmacokinetic properties for oral dosing. Collectively, this work establishes periplasmic targeted protein degradation as a promising new mechanism for combating {beta}-lactamase resistance.