Biology subjects

Mulholland, A. J.

Publications and source records attributed to Mulholland, A. J..

At least 19 recordsLinked to original sources

bioRxiv2026

De Novo Design of Flavoproteins with Controlled Excited State Dynamics

Designing photochemical properties in proteins is a frontier challenge in biomolecular design, which, when realised, will open new avenues in catalysis and biomolecular electronics. Here we demonstrate an integrated computational design framework, supported by ultrafast spectroscopy, combining protein design, quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) and electronic structure calculations, to control flavin excited states in de novo proteins. Iterative design yields FC-4D2, a flavocytochrome with covalently bound flavin and non-covalently bound heme B and Fl-4D2, where the heme-binding site is removed. Both proteins are highly thermostable, and access all physiologically relevant flavin redox and protonation states, while high-resolution X-ray crystallography confirms the near-[A]ngstrom-level accuracy in design. Incorporating QM/MM MD and linear-response time-dependent density functional theory (LR-TDDFT) into the design workflow enables engineering of a flavin-tryptophan charge-transfer variant, Fl-4D2CT, that modifies the flavin absorption spectrum, substantially alters its redox properties and suppresses fluorescence. Ultrafast studies reveal how protein design reshapes the excited state relaxation timescale and mechanism. Our study establishes a modular strategy for programming flavin photochemistry in designed proteins.

biophysics↗

bioRxiv2026

Cancer-associated mutations reconfigure dynamical responses in EGFR kinase

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays a fundamental role in regulating cellular proliferation, survival, and differentiation. EGFR's kinase domain catalyses the autophosphorylation that drives downstream signalling and is a known hotspot for cancer-associated mutations. Such substitutions often enhance receptor activation, driving oncogenesis and therapeutic resistance, with major implications for prognosis and treatment choice; consequently, a detailed understanding of this domain's functional dynamics, and how mutations reshape them, is essential. Here, we apply dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to probe how changes at the ATP-binding site are transmitted throughout the kinase domain, ultimately shaping the behaviour of distal regions in the wild-type protein and in two of the most clinically observed non-small cell lung cancer variants, L858R and L858R+T790M. D-NEMD reveals previously uncharacterised communication networks linking the ATP pocket to key functional motifs, including the P-loop, C-helix, hinge region, activation loop, and F-helix, showing the intricate allosteric connectivity within this domain. Notably, numerous cancer-related mutation sites, despite being distant from the ATP site, map onto or lie close to these communication pathways, indicating that D-NEMD simulations can identify functionally relevant allosteric positions and potential mutation sites in EGFR. Our simulations further reveal that the two variants rewire EGFR's internal signal propagation network in distinct ways: L858R diminishes the transmission of structural changes to distal regions of the C-lobe, whereas introducing T790M in addition to L858R partially restores wild-type-like behaviour through compensatory rerouting of early-stage dynamical pathways.

biochemistry↗

bioRxiv2026

Machine learned potentials with electrostatic embedding accurately capture Kemp eliminase reactivity

Kemp elimination has become a benchmark for de novo enzyme design due to its simplicity and detailed mechanistic understanding but accurate barrier calculations are required to understand the difference in activity between designed Kemp eliminases. QM/MM MD simulations are capable of calculating reaction barriers, but are limited by a cost/accuracy tradeoff in which the most accurate methods are too computationally expensive for extensive screening as would be required in a prospective enzyme design campaign. Recent advances in embedded ML/MM simulations using the electrostatic machine learning embedding (EMLE) method have enabled transferable potentials for ML/MM MD simulations with low computational cost but QM-level accuracy. Here, we trained a MACE MLIP and EMLE embedding model for fast and accurate simulations of the enzymatic, base-catalysed Kemp elimination of 6-nitro benzisoxazole. Applying our EMLE ML/MM scheme to a designed Kemp eliminase and its evolved counterpart captured the {approx}4 kcal/mol difference in barrier between the two observed in experiment. Furthermore, training was done using structures generated from simulations of a single variant and was applied to the second variant, achieving near-experimental barriers, with no further finetuning or computational overhead. Thus, this work establishes EMLE-based ML/MM MD simulations as a potential route for fast and accurate assessment of barriers in computational screening for enzyme design.

biochemistry↗

bioRxiv2026

Evolution promotes transition-state-like conformations with enhanced basicity and electric fields in the substrate complex of a designer enzyme

Enzymes can be computationally designed for an increasing range of reactions, yet the catalytic efficiencies achieved are typically well below those of natural biocatalysts. Directed evolution can narrow this gap, often improving activity through mutations with non-obvious effects on catalysis. Here, we dissect how directed evolution improved a de novo designed Kemp eliminase by comparing the ground-state and transition-state ensembles of the designed and evolved variants. Extensive molecular dynamics simulations and QM/MM reaction barrier calculations identify subtle but important differences that modulate reactivity. Simulations show that reaction proceeds via a specific transition-state-like conformation, characterized by desolvation of the catalytic base and an organized active-site electric field. Barriers correlate strongly with the reaction energy of proton transfer, indicating that desolvating the catalytic base in the ligand-bound complex raises its proton affinity and thereby lowers the barrier. Evolution enriches the ground-state ensemble in reactive conformations, minimizing the reorganization required to reach the transition state. After evolution, formation of the transition-state-like conformation causes less disruption to the protein, which maintains coordinated motions. This is consistent with the emergence of a negative activation heat capacity with evolution. Efficient catalysis requires a ground-state ensemble that resembles the transition state, an objective that enzyme design should pursue alongside geometric and electrostatic complementarity for the transition state.

biochemistry↗

bioRxiv2026

Redox Control of S-sulfocysteine Formation in Adenosine Phosphosulfate Reductase

Sulfur assimilation fuels bacterial growth by supplying the reduced sulfur required for the biosynthesis of sulfur-containing biomolecules. Adenosine 5'-phosphosulfate reductase (APSR) catalyzes the first reductive step of this pathway, converting adenosine 5'-phosphosulfate (APS) to adenosine monophosphate. This reaction proceeds through nucleophilic attack by catalytic C256, located in the flexible C-terminal tail, on the sulfur atom of APS, forming a thiosulfonate enzyme intermediate. Here, we investigate this reaction in APSR from Pseudomonas aeruginosa, an opportunistic pathogen associated with severe infections, particularly in patients with cystic fibrosis. APSR contains an iron-sulfur [4Fe-4S] cluster, which participates in redox steps of the reaction. Here, we show that the redox state of the iron-sulfur cluster also controls the catalytic step. Multiscale molecular simulations investigate how oxidized and reduced cluster states affect APS binding, active site organization, and the nucleophilic attack step. Molecular dynamics (MD) simulations show that the oxidized [4Fe-4S]2+; cluster stabilizes substrate interactions and the conformation of the C-terminus, facilitating a catalytically productive orientation of C256. The activation barrier of 17.7 {+/-} 1.7 kcal mol-1 from quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations at the B3LYP-D3(BJ)/6-31G(d) level of theory is in good agreement with the experimental kinetics. The redox state of the iron-sulfur cluster shows its role in modulating the conformation of conserved K144, which is important for transition state stabilization in the nucleophilic attack. These findings illuminate the mechanism of this P. aeruginosa target and provide broader insight into the roles of iron-sulfur clusters in controlling enzyme reactivity.

biochemistry↗

bioRxiv2026

A transition state-like acylenzyme conformation distinguishes carbapenemase activity in class A β-lactamases

Carbapenems are the most potent {beta}-lactams, key antibiotics for healthcare-associated infections by Gram-negative bacteria and evade hydrolysis by most {beta}-lactamases, but are increasingly threatened by emergence of enzymes exhibiting hydrolytic activity towards them. Of the four recognised {beta}-lactamase subclasses, class A (active-site serine enzymes that hydrolyse {beta}-lactams via a covalent acylenzyme intermediate) is the most widely disseminated and, while the majority of such enzymes react with carbapenems to form long-lasting acylenzyme complexes, several possess carbapenem-hydrolyzing activity (carbapenemases). Here, we investigate the basis for these differences in a panel of class A {beta}-lactamases using molecular dynamics (MD) simulations of the respective acylenzyme complexes and tetrahedral intermediates (TI). The simulations reveal multiple features associated with catalytic activity across the spectrum of enzymes tested, including more extensive interactions of the carbapenem acylenzyme carbonyl and generally increased lifetimes of active site water molecules positioned for deacylation. Analysis of the dynamic trajectories shows carbapenemases to have reduced root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI, that are not limited to the active site, indicating that the acylenzyme complex is pre-organised for reaction in carbapenemases but not in carbapenem-inhibited enzymes. Similarly, Principal Component Analysis (PCA) of acylenzyme and TI dynamics shows greater overlap between the two states in carbapenemases, providing further evidence for acylenzyme pre-organisation. Such simulations may represent an effective computational assay able to identify enzymes with carbapenemase activity at relatively modest computational cost.

biochemistry↗

bioRxiv2026

Enhanced Target Binding by Leritrelvir Restores Dimerization of Mpro Mutants and Mitigates Drug Resistance

The SARS-CoV-2 main protease (Mpro) has been a major target of antiviral drug development, leading to the development of inhibitors such as nirmatrelvir, the antiviral component of the COVID-19 drug Paxlovid. However, resistance-associated mutations that reduce the efficacy of current Mpro inhibitors, particularly nirmatrelvir, have emerged. Here, we evaluated the inhibitory activity of leritrelvir (RAY1216), an Mpro inhibitor approved in China for COVID-19 monotherapy, against a panel of Mpro variants carrying mutations at 12 resistance-associated residues distributed across four catalytic subsites. Using integrated biochemical, biophysical, structural, and cellular analyses, we demonstrate that leritrelvir retains stronger inhibitory activity against most tested resistant mutants compared with nirmatrelvir. Most of the tested mutations promote Mpro dimer dissociation, with E166V showing a particularly pronounced effect and markedly compromising nirmatrelvir binding. In contrast, thermal shift and size-exclusion chromatography assays demonstrate that leritrelvir binding restores dimerization of these Mpro mutants. Sixteen high-resolution crystal structures reveal that leritrelvir binding re-instates key dimer-interface interactions disrupted by resistance mutations. Mini-replicon assays further confirm leritrelvir to possess enhanced cellular antiviral efficacy compared with nirmatrelvir. Our findings indicate that tighter leritrelvir binding enables more effective inhibition of dissociation-prone Mpro mutants than nirmatrelvir, supporting its use as a more resilient antiviral agent for SARS-CoV-2 treatment.

molecular biology↗

bioRxiv2026

Porphyrin driven redox tuning in structurally defined de novo heme proteins

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

biochemistry↗

bioRxiv2026

Acyl-enzyme dynamics, tautomerisation and hydration regulate turnover of carbapenem antibiotics by the OXA-48 β-lactamase

OXA-48 is a globally disseminated class D serine {beta}-lactamase that efficiently confers resistance to a range of {beta}-lactam antibiotics, including carbapenems, the most potent such agents versus Enterobacterales (Escherichia coli and relatives). Here we characterise the interactions of OXA-48 with the acyl-enzyme complex intermediates formed on its reaction with the carbapenems meropenem and ertapenem using X-ray crystallography and molecular dynamics (MD) simulations. X-ray crystal structures identify acyl-enzymes in both the {Delta}1-imine and {Delta}2-enamine pyrroline tautomeric forms. MD simulations show the epimeric {Delta}2 tautomers of meropenem and ertapenem to more frequently adopt binding poses competent for hydrolysis, i.e. with an appropriate orientation of the carbapenem 6-hydroxyethyl group and positioning of the water molecule required for deacylation; the results indicate that the {Delta}2 tautomers are preferred for deacylation over the {Delta}1-tautomer. MD simulations based on the crystal structures show that, compared to OXA-48, acyl-enzyme complexes of OXA-519 (a natural OXA-48 variant with a single Val120Leu substitution adjacent to the catalytic general base) more frequently sampled conformations favouring hydrolysis, or formation of the alternative {beta}-lactone deacylation product. MD simulations of complexes derived from quantum mechanics/molecular mechanics (QM/MM) simulations show the meropenem-derived {beta}-lactone product is better retained in the OXA-48 active site than hydrolysed meropenem, consistent with reversible {beta}-lactone formation. Overall, our results demonstrate how acyl-enzyme tautomerisation, dynamics and hydration collectively modulate degradation of 1{beta}-methyl carbapenems by class D {beta}-lactamases of the OXA-48 group, and how subtle changes in active site structure potentiate such effects in the OXA-519 variant.

biochemistry↗

bioRxiv2026

Hydrogen-bonding changes cause differences in imipenem breakdown activity in OXA-48 variants

The {beta}-lactamase OXA-48 efficiently hydrolyses carbapenem antibiotics, especially imipenem. Carbapenem resistance is a rising clinical concern, and is frequently associated with OXA-48 and its variants. OXA-48 variants carrying different mutations in the {beta}5-{beta}6 loop differ in hydrolytic activity towards imipenem. OXA-517 has a higher KM, but similar kcat for imipenem hydrolysis, compared to OXA-48, whereas OXA-163 and -405, which have similar mutations in the {beta}5-{beta}6 loop, are less active. Multiscale simulations (using quantum mechanics/molecular mechanics, QM/MM) of deacylation of the respective imipenem acylenzymes show this to be most efficient when the deacylating water (DW) acts as a hydrogen bond (H-bond) donor to imipenem, and the carboxylated Lys73 base is less hydrated. Calculated barriers for deacylation correlate very well with experimental data, but for OXA-163 and -405 only when DW acts as a H-bond acceptor. Dynamics simulations of imipenem acylenzyme complexes show that mutations in the {beta}5-{beta}6 loop change the active site H-bond network. In OXA-48, the DW H-bonding pattern linked to high activity is more frequently sampled, and in OXA-517 it is stabilised through H-bonding to Thr213; explaining the higher kcat values compared to OXA-163 and -405, where this is not the case. Furthermore, simulations of non-covalent imipenem complexes indicate that increased KM for OXA-517 is linked to lower binding affinity, caused by repositioning of bound imipenem. Our work identifies the molecular basis for differences in imipenem hydrolytic activity between OXA-48 variants, offering detailed insights into how active site interactions alter the dynamics and reaction efficiencies related to antibiotic resistance.

biochemistry↗

bioRxiv2025

Correlated dynamics, reactive conformations and non-Arrhenius behaviour in the temperature-dependence of enzyme activity: triosephosphate isomerase

Heat capacity changes are increasingly recognised as crucial for understanding the temperature dependence of enzyme-catalysed reaction rates. Here, we combine experiments and molecular dynamics simulations to investigate triosephosphate isomerase (TIM, TPI) from psychrophilic, mesophilic and thermophilic organisms. Kinetic data show clear curvature in rate-temperature plots, particularly for the cold-adapted enzyme, independent of unfolding. This non-Arrhenius behaviour is accounted for by Macromolecular Rate Theory (MMRT), implying an activation heat capacity, largest for the psychrophile. Simulations reveal the molecular origins of these differences, showing significant conformational and dynamical changes between reactant and transition states in the cold-adapted enzyme, with a smaller activation heat capacity in the mesophilic enzyme, and near-zero for the thermophile. Transition-state-like conformations and reorganized correlated dynamics underlie these adaptations. These results demonstrate that dynamical differences between crucial states along the reaction pathway play a key role in determining the optimum temperature of activity for this archetypal enzyme.

biochemistry↗

bioRxiv2025

Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

{beta}-Lactamases catalyze {beta}-lactam antibiotic hydrolysis and are important contributors to bacterial antimicrobial resistance; {beta}-lactamase inhibitors are widely used to overcome {beta}-lactamase-mediated antibiotic resistance. Nucleophilic serine {beta}-lactamases (SBLs) react with their substrates and clinically available inhibitors via a covalent reaction to give complexes which can undergo further reaction. Using room temperature drop on fixed target serial crystallography, where ligands are rapidly mixed with microcrystals, and classical single-crystal crystallography at cryogenic temperatures, we investigate the reversible covalent reaction of the SBL CTX-M-15 with the diazobicyclooctane inhibitor avibactam. We observe avibactam covalently reacted (ring-opened) with the nucleophilic Ser70, at timepoints from 80 ms to minutes (room temperature) and hours (100 K). These crystallographic data reveal time-dependent movement of the avibactam carbamoyl complex, from 1.3 s onwards, that has implications for the 5-exo-trig recyclization mechanism that determines inhibitor reformation. Combined with molecular dynamics simulations and quantum mechanics calculations at the density functional theory level, the results show that in the first seconds of the reaction the avibactam N-sulfate nitrogen is poorly positioned for recyclization. This subsequently equilibrates after 10 s to a stable endpoint that is in a conformation potentially primed to initiate recyclization through attack of the N-sulfate nitrogen on the carbamoyl carbon. These results further demonstrate the capacity of room-temperature serial crystallography to capture time-resolved changes in ligand conformation at an enzyme active site, complementing discrete classical cryo-crystallography. These data inform on ligand dynamics and the stereoelectronics of diazobicyclooctane inhibition, aiding drug discovery efforts to develop inhibitors of nucleophilic enzymes.

biochemistry↗

bioRxiv2025

A dynamical-nonequilibrium molecular dynamics (D-NEMD) alanine scanning approach for identifying allosteric positions in proteins

Allosteric effects are widespread in proteins, but predicting the impact of sequence substitutions at positions distant from ligand-binding or enzyme active sites remains challenging, as their effects are mediated through complex dynamical networks. Pinpointing such positions experimentally is labour-intensive and low throughput. Equilibrium molecular dynamics (MD) simulations are useful: analysis methods can identify functional distal sites and networks, while free energy simulations can predict effects on stability or binding, given sufficient sampling; such simulations are typically time-consuming, requiring extensive simulation of each individual mutant. Here, we introduce a dynamical-nonequilibrium MD (D-NEMD) protocol using alanine substitutions as targeted perturbations to identify distal positions that influence enzyme function. Using the well characterised class A {beta}-lactamase KPC-2 as a test system, we show that D-NEMD distinguishes functional from non-functional sites based on whether substitutions generate structured, long-range responses that reach the active site. KPC-2 is clinically important due to its broad substrate spectrum and the emergence of resistance-associated point mutations, many of which act through non-local effects on catalytic residues. Alanine substitutions at positions 179 and 164, which disrupt the {Omega}-loop salt bridge, trigger persistent structural responses propagating through the protein and reach both catalytic residues and the oxyanion-hole backbone. Likewise, alanine substitution at position 220 elicits pronounced responses extending to the active-site {beta}-sheet and key loops, consistent with its known role in substrate specificity. In contrast, substitution at position 276--mutations of which have negligible kinetic impact--produces only local displacements with minimal propagation and no effect on catalytically relevant regions. These differential response patterns align with experimentally observed resistance phenotypes. D-NEMD therefore provides a fast, generalisable, and predictive approach for identifying allosterically connected distal positions. The protocol complements equilibrium MD, is straightforward to implement, and offers a tractable route for prioritising candidate sites for mechanistic study or future mutational scanning efforts. Statement of significanceWe introduce a dynamical-nonequilibrium MD (D-NEMD) protocol that uses alanine substitutions as the perturbation to identify residues, particularly those distal to an active or binding site, that modulate biological activity. We test this approach on the clinically important {beta}-lactamase KPC-2: it distinguishes positions that elicit long-range responses that reach catalytic residues from another with only local effects, in agreement with experiment. The D-NEMD approach allows for tests of statistical robustness of responses to mutation, enabling prioritisation of candidate residues for experimental mutation. This should aid in identifying distal sites that may influence enzyme activity or serve other allosteric roles. The approach is generalisable to other proteins of biomedical and biotechnological interest.

biochemistry↗

bioRxiv2025

pH-induced structural changes in SARS-CoV-2 spike variants

pH critically influences SARS-CoV-2 infectivity and stability by, for example, triggering pH-dependent conformational changes in the spike glycoprotein that can facilitate membrane fusion and viral entry into host cells. Using the emerging dynamical nonequilibrium molecular dynamics (D-NEMD) simulations approach, we investigated how biologically relevant pH shifts affect the functional dynamics of the fully glycosylated spike from the ancestral, Delta, and Omicron BA.1 variants of concern. For this, over 1100 nonequilibrium simulations were conducted to capture the pH-induced structural and dynamic changes that occur following transitions from physiological to acidic and alkaline conditions, with the former mimicking the low pH environment within endosomes, and the latter the high pH conditions accessible to nascent exhaled aerosols. D-NEMD reveals that pH changes trigger distinct, variant-specific conformational responses in key regions of the spike, including the receptor binding domain (RBD), fusion peptide proximal region (FPPR), and C-terminal domain (CTD). The ancestral spike shows broad pH sensitivity, characterised by directional motions and region-specific structural rearrangements that depend on the pH conditions. The spike of the Delta variant displays increased reactivity to alkaline pH, potentially explaining its reduced stability in alkaline aerosols. The spike of Omicron BA.1, in contrast, responds strongly to acidic conditions with spontaneous RBD opening and pronounced structural rearrangements in the FPPR and CTD. This behaviour aligns with this variants preference for endosomal entry and reduced reliance on TMPRSS2-mediated fusion. The Omicron BA.1 spike also shows increased resilience to alkaline pH, suggesting greater environmental stability. Our findings further emphasise the key role of glycans in spike activation, with glycan N234 stabilising the RBD "up" conformation during pH-induced transitions in Omicron under acidic conditions. These insights, together, highlight pH as a potential evolutionary pressure for SARS-CoV-2 and underscore the importance of glycosylation and environmental pH variability in shaping the behaviour of viral fusion proteins.

biochemistry↗

bioRxiv2025

Effective sequence-to-expression prediction for membrane proteins using machine learning and computational protein design

The recombinant expression of integral membrane proteins is notoriously challenging. One way to address this challenge is via computational genotype-to-phenotype models that determine how particular sequence features correlate with protein expression levels. However, the potential of such approaches is yet to be fully realised, at least partly because so few expression datasets are available. Here, we study the sequence-to-expression relationships of a library of 12,248 variants of a specific membrane protein derived from combinatorial computational design. The major advantage of this approach lies in the controlled sequence diversity explored in design, making this new dataset directly compatible with lightweight off-the-shelf bioinformatic tools. The expression phenotype of the entire library is assessed in the widely-used recombinant host Escherichia coli. We employed a relatively small dataset of [~]2000 phenotyped sequences to train a sequence-to-expression predictor using supervised machine learning, which achieved high classification accuracy on held-out test sequences. This model was then used to infer the expression of >10,000 unmeasured sequences, and validation of the top predictions of both high and low expressers achieved 100% success rate. Using tools from explainable AI, we identified specific sequence positions and substitutions that are most important in dictating cellular expression levels. This analysis was validated by model-guided protein engineering that achieved an 8-fold increase in the purification yield of a poorly-expressing variant. Our results show that, at least for this controlled dataset, straightforward and interpretable machine learning can reveal the intrinsic sequence code for membrane protein expression.

bioengineering↗

bioRxiv2025

Mechanistic Basis for Inhibition of the Extended Spectrum Class A b-Lactamase GES-1 by Tazobactam and Enmetazobactam

Expression of {beta}-lactamases is the primary form of {beta}-lactam antibiotic resistance in Gram-negative bacteria. Enmetazobactam is a penicillanic acid sulfone (PAS) that inhibits extended spectrum {beta}-lactamases (ESBLs) by forming an acyl-enzyme complex that eventually breaks down to an irreversible lysinoalanine crosslink. In contrast, enmetazobactam inhibits the class A carbapenemase KPC-2 via an acyl-enzyme that does not lead to lysinoalanine crosslink formation. This difference correlates with greater inhibitory potency of enmetazobactam against class A ESBLs, compared to carbapenemases. The GES enzymes, unlike other class A {beta}-lactamase families, show progression from carbapenem-inhibited to carbapenem-hydrolysing phenotypes through single point mutations. We present crystal structures of GES-1, a globally disseminated ESBL, as the enmetazobactam- and tazobactam-derived acyl-enzymes. The complexes differ in the identities of their respective covalent adducts, with the catalytic Ser70 acylated by a 214 Da enmetazobactam-derived fragment, whereas tazobactam has fragmented to a 70 Da aldehyde. The tautomeric form of the enmetazobactam-derived ligand is verified by high-level QM/MM calculations, revealing the trans-enamine as the most thermodynamically stable tautomer, that adopts an optimised conformation that best matches the experimentally observed electron density appended to the side chain oxygen of Ser70. In contrast to previous findings for the ESBL CTX-M-15, mass spectrometry provides no evidence for lysinoalanine crosslink formation on reaction of GES-1 with (enme)tazobactam, providing further evidence that PAS inhibitors inhibit different class A {beta}-lactamases by different mechanisms. This work reveals new details of the basis for PAS inhibition of diverse {beta}-lactamases, and will guide development of future {beta}-lactamase inhibitors.

biochemistry↗

bioRxiv2025

AI.zymes - A modular platform for evolutionary enzyme design

The ability to create new-to-nature enzymes would substantially advance bioengineering, medicine, and the chemical industry. Despite recent breakthroughs in protein design and structure prediction, designing biocatalysts with activities rivaling those of natural enzymes remains challenging. Here, we present AI.zymes, a modular platform integrating cutting-edge protein engineering algorithms within an evolutionary framework. By combining programs such as Rosetta, ESMFold, ProteinMPNN, and FieldTools in iterative rounds of design and selection, AI.zymes optimizes a broad range of catalytically relevant properties, such as transition state affinity and protein stability. Notably, AI.zymes can also improve properties that are not targeted by the employed design algorithms. For instance, AI.zymes enhanced electrostatic catalysis by iteratively selecting variants with stronger catalytic electric fields. AI.zymes was benchmarked by improving the promiscuous Kemp eliminase activity of ketosteroid isomerase, yielding a 7.7-fold increase in activity after zero-shot experimental validation of only 7 variants. Due to its modularity, AI.zymes can readily incorporate emerging design algorithms, paving the way for a unifying framework for enzyme design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/633707v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f2ac2eorg.highwire.dtl.DTLVardef@61b2aorg.highwire.dtl.DTLVardef@e176edorg.highwire.dtl.DTLVardef@46d692_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Structure and dynamics of Burkholderia pseudomallei OXA-57, a distinctive low-efficiency class D β-lactamase with carbapenem-hydrolyzing activity

The Gram-negative bacterium Burkholderia pseudomallei causes the severe disease melioidosis. {beta}-Lactams, including carbapenems, are the primary treatment, but are susceptible to chromosomal {beta}-lactamases, including the class D enzyme OXA-57. Here we show recombinant OXA-57 is active towards penicillins and first-generation cephalosporins, slowly hydrolyzes carbapenems including imipenem and meropenem, but is inactive towards oxyimino-cephalosporins (e.g., ceftazidime). Unlike many OXA enzymes, OXA-57 is sensitive to the mechanism-based inhibitor clavulanic acid, but less so to the diazabicyclooctane avibactam and not to the cyclic boronate vaborbactam. Crystal structures of covalent OXA-57:avibactam and OXA-57:meropenem complexes reveal limited hydrogen- bonding interactions with bound ligands. In molecular dynamics simulations, bound meropenem is mobile, while the water necessary for deacylation has only limited active site access. These observations are consistent with the low level of meropenem turnover, supporting proposals that OXA {beta}-lactamases generally possess limited carbapenemase activity, and highlight the potential importance of OXA-57 in B. pseudomallei {beta}-lactam resistance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/630153v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@d635d7org.highwire.dtl.DTLVardef@168d67borg.highwire.dtl.DTLVardef@1d95733org.highwire.dtl.DTLVardef@1ebeabb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗