A curated list of targeted optimized promiscuous ketoreductases (TOP-K).

Enzymes are either specific or promiscuous catalysts in nature. The latter is portrayed by protein families like CYP450Es, Aldo-ketoreductases and short/medium-chain dehydrogenases which participate in detoxification or secondary metabolite production. However, enzymes are evolutionarily 'blind' to an ever-increasing synthetic substrate library. Industries and laboratories have circumvented this by high-throughput screening or site-specific engineering to synthesize the product of interest. However, this paradigm entails cost and time-intensive one-enzyme, one-substrate catalysis model. One of the superfamilies regularly used for chiral alcohol synthesis are short-chain dehydrogenases/reductases (SDRs). Our objective is to determine a superset of promiscuous SDRs that can catalyze multiple ketones. They are typically classified into shorter 'Classical' and longer 'Extended' type ketoreductases. However, current analysis of modelled SDRs reveals a length-independent conserved N-terminus Rossmann-fold and a variable substrate-binding C-terminus substrate-binding region for both categories. The latter is recognized to influence the enzyme's flexibility and substrate promiscuity and we hypothesize these properties are directly linked with each other. We tested this by catalyzing ketone intermediates with the essential and specific enzyme: FabG_E, as well as non-essential SDRs such as UcpA and IdnO. The experimental results confirmed this biochemical-biophysical association, making it an interesting filter for ascertaining promiscuous enzymes. Hence, we created a dataset of physicochemical properties derived from the protein sequences and employed machine learning algorithms to examine potential candidates. This resulted in 24 targeted optimized ketoreductases (TOP-K) from 81 014 members. The experimental validation of select TOP-Ks demonstrated the correlation between the C-terminal lid-loop structure, enzyme flexibility and turnover rate on pro-pharmaceutical substrates.

A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%. CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

Myelin basic protein cleaves cell adhesion molecule L1 and promotes neuritogenesis and cell survival.

The cell adhesion molecule L1 is a Lewis(x)-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis(x)-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg(687) yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system.

BWC0977, a broad-spectrum antibacterial clinical candidate to treat multidrug resistant infections.

The global crisis of antimicrobial resistance (AMR) necessitates the development of broad-spectrum antibacterial drugs effective against multi-drug resistant (MDR) pathogens. BWC0977, a Novel Bacterial Topoisomerase Inhibitor (NBTI) selectively inhibits bacterial DNA replication via inhibition of DNA gyrase and topoisomerase IV. BWC0977 exhibited a minimum inhibitory concentration (MIC90) of 0.03-2 µg/mL against a global panel of MDR Gram-negative bacteria including Enterobacterales and non-fermenters, Gram-positive bacteria, anaerobes and biothreat pathogens. BWC0977 retains activity against isolates resistant to fluoroquinolones (FQs), carbapenems and colistin and demonstrates efficacy against multiple pathogens in two rodent species with significantly higher drug levels in the epithelial lining fluid of infected lungs. In healthy volunteers, single-ascending doses of BWC0977 administered intravenously ( https://clinicaltrials.gov/study/NCT05088421 ) was found to be safe, well tolerated (primary endpoint) and achieved dose-proportional exposures (secondary endpoint) consistent with modelled data from preclinical studies. Here, we show that BWC0977 has the potential to treat a range of critical-care infections including MDR bacterial pneumonias.

Validated In Silico Population Model of Escherichia coli.

Flux balance analysis (FBA) and ordinary differential equation models have been instrumental in depicting the metabolic functioning of a cell. Nevertheless, they demonstrate a population's average behavior (summation of individuals), thereby portraying homogeneity. However, living organisms such as Escherichia coli contain more biochemical reactions than engaging metabolites, making them an underdetermined and degenerate system. This results in a heterogeneous population with varying metabolic patterns. We have formulated a population systems biology model that predicts this degeneracy by emulating a diverse metabolic makeup with unique biochemical signatures. The model mimics the universally accepted experimental view that a subpopulation of bacteria, even under normal growth conditions, renders a unique biochemical state, leading to the synthesis of metabolites and persister progenitors of antibiotic resistance and biofilms. We validate the platform's predictions by producing commercially important heterologous (isobutanol) and homologous (shikimate) metabolites. The predicted fluxes are tested in vitro resulting in 32- and 42-fold increased product of isobutanol and shikimate, respectively. Moreover, we authenticate the platform by mimicking a bacterial population in the presence of glyphosate, a metabolic pathway inhibitor. Here, we observe a fraction of subsisting persisters despite inhibition, thus affirming the signature of a heterogeneous populace. The platform has multiple uses based on the disposition of the user.

Validated In Silico Model for Biofilm Formation in Escherichia coli.

Using Escherichia coli as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on ∼300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic E. coli were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae.

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Azaindole Based Potentiator of Antibiotics against Gram-Negative Bacteria.

We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane's integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.

Lewis(x) and alpha2,3-sialyl glycans and their receptors TAG-1, Contactin, and L1 mediate CD24-dependent neurite outgrowth.

Although carbohydrates have been implicated in cell interactions in the nervous system, the molecular bases of their functions have remained largely obscure. Here, we show that promotion or inhibition of neurite outgrowth of cerebellar or dorsal root ganglion neurons, respectively, induced by the mucin-type adhesion molecule CD24 depends on alpha2,3-linked sialic acid and Lewis(x) present on glia-specific CD24 glycoforms. Alpha2,3-sialyl residues of CD24 bind to a structural motif in the first fibronectin type III domain of the adhesion molecule L1. Following the observation that the adhesion molecules TAG-1 and Contactin show sequence homologies with fucose-specific lectins, we obtained evidence that TAG-1 and Contactin mediate Lewis(x)-dependent CD24-induced effects on neurite outgrowth. Thus, L1, TAG-1, and Contactin function as lectin-like neuronal receptors. Their cis interactions with neighboring adhesion molecules, e.g., Caspr1 and Caspr2, and with their triggered signal transduction pathways elicit cell type-specific promotion or inhibition of neurite outgrowth induced by glial CD24 in a glycan-dependent trans interaction.

Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy.

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue.

The Mycobacterium tuberculosis murG gene, Rv2153, was expressed in Escherichia coli murG(Ts) strain OV58 on a plasmid under the control of the arabinose-inducible araBAD promoter. Mycobacterium tuberculosis murG rescued the growth of E. coli murG(Ts) at the nonpermissive temperature: transformants were only obtained in the presence of 0.2% arabinose at 42 °C, and their growth rate was dependent on arabinose concentrations. However, no MurG activity was detected in membranes from the transformant grown in arabinose at 42 °C, while MraY activity was normal. This observation led to the development of a membrane-based scintillation proximity assay for exogenous sources of MurG. Addition of purified E. coli MurG resulted in the reconstitution of MurG and peptidoglycan synthesis in these membranes. MurG is an attractive target for drug discovery, but assays to measure the activity of purified MurG are challenging. This presents an easy method to measure the activity of exogenous sources of MurG for structure-activity studies of mutant MurG proteins. It can also be used to compare the activity of, or effect of inhibitors on, MurG from other bacterial species.

Identification and validation of a Lewis x glycomimetic peptide.

Glycans play important roles in regulating cell recognition and interactions to fine tune development, and synaptic plasticity and regeneration in the adult nervous system. The spatial and temporal expression pattern of Lewis(x) (a terminal trisaccharide epitope characterized by alpha1,3-fucosyl-N-acetyl-lactosamine) in the nervous system indicates an important role of this epitope in neurogenesis and brain development. Localization of Lewis(x) in the proliferative subventricular zone of the developing nervous system and also its expression on stem cells of the adult nervous system suggests a role in neurogenesis and hence regeneration. To provide an alternative tool to elucidate the functional roles of Lewis(x), we screened a random peptide phage library against a Lewis(x)-specific antibody to identify a Lewis(x) glycomimetic peptide. We identified a peptide that specifically bound to the Lewis(x)-specific antibody and this binding could be competed by the Lewis(x) glycan. Different aspects of the Lewis(x) glycomimetic peptide were investigated by introducing it in in vitro assays measuring neurite outgrowth and in in vivo assays to determine its efficacy in regeneration of peripheral nerve and spinal cord after injury in adult mice. In vitro, neurite outgrowth triggered by the Lewis(x-)carrying adhesion molecule CD24 was abolished alike by the Lewis(x) glycan and the glycomimetic peptide, while no influence of the glycomimetic peptide was seen in regeneration. Our results validate the use of Lewis(x) glycomimetic peptide as a functionally equivalent structure to the Lewis(x) glycan.