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Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we isolated HsKYNase_66 through an unusual evolutionary trajectory, having a 410-fold increase in the k cat /K M for kynurenine (KYN) and reverse substrate selectivity relative to HsKYNase. Here, by following a different evolutionary trajectory we generate a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a "generalist" that accepts 3'-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis reveals that while the evolution of HsKYNase_66 is accompanied by a change in the rate-determining step of the reactions, HsKYNase_93D9 retains the same catalytic mechanism as HsKYNase. HDX-MS shows that the conformational dynamics of the two enzymes are markedly different and distinct from ortholog prokaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship between evolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species., (© 2024. The Author(s).)

In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm 3 -bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4 %. Moreover, using the scFv column of R2-47, 90.7 % of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67 % of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E. coli cell lysate by one-step affinity purification, and 89.7 % of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >10 9 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution., Competing Interests: Competing interests J.B., C.S.K., N.M., W.C.L., G.G., and E.S. are inventors on intellectual property related to this work, including the active patent US9975959B2 and the pending patents US20190350975A1 and US20210207110A1, which are assigned to the Board of Regents of the University of Texas System. G.G., E.S., X.M.Z., K.M., S.C., and M.M. have equity interest in Ikena Oncology, a company pursuing the commercial development of this technology. J.B., C.S.K., C.L., and E.S. have consulted for Ikena Oncology (previously Kyn Therapeutics). The remaining authors declare no competing interests.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre–steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5′-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli , whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N'-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes., Competing Interests: CSK, CS, GA and SR were employed by Aeglea Biotherapeutics., (Copyright © 2021 Karamitros, Somody, Agnello and Rowlinson.)

Kynureninases (KYNases) are enzymes that play a key role in tryptophan catabolism through the degradation of intermediate kynurenine and 3'-hydroxy-kynurenine metabolites (KYN and OH-KYN, respectively). Bacterial KYNases exhibit high catalytic efficiency toward KYN and moderate activity toward OH-KYN, whereas animal KYNases are highly selective for OH-KYN, exhibiting only minimal activity toward the smaller KYN substrate. These differences reflect divergent pathways for KYN and OH-KYN utilization in the respective kingdoms. We examined the Homo sapiens and Pseudomonas fluorescens KYNases (HsKYNase and PfKYNase respectively) using pre-steady-state and hydrogen-deuterium exchange mass spectrometry (HDX-MS) methodologies. We discovered that the activity of HsKYNase critically depends on formation of hydrogen bonds with the hydroxyl group of OH-KYN to stabilize the entire active site and allow productive substrate turnover. With the preferred OH-KYN substrate, stabilization is observed at the substrate-binding site and the region surrounding the PLP cofactor. With the nonpreferred KYN substrate, less stabilization occurs, revealing a direct correlation with activity. This correlation holds true for PfKYNases; however there is only a modest stabilization at the substrate-binding site, suggesting that substrate discrimination is simply achieved by steric hindrance. We speculate that eukaryotic KYNases use dynamic mobility as a mechanism of substrate specificity to commit OH-KYN to nicotinamide synthesis and avoid futile hydrolysis of KYN. These findings have important ramifications for the engineering of HsKynase with high KYN activity as required for clinical applications in cancer immunotherapy. Our study shows how homologous enzymes with conserved active sites can use dynamics to discriminate between two highly similar substrates.

Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for l-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. We developed three expression vectors to produce and localize l-asparaginase (l-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T m ) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T m compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction., Competing Interests: Declaration of Interests JJG is an unpaid board member of the Rosetta Commons. Under institutional participation agreements between the University of Washington, acting on behalf of the Rosetta Commons, Johns Hopkins University may be entitled to a portion of revenue received on licensing Rosetta software including programs described here. As a member of the Scientific Advisory Board of Cyrus Biotechnology, JJG is granted stock options. Cyrus Biotechnology distributes the Rosetta software, which may include methods described in this article.

In the present study, we report the effect of four different soluble additives (sucrose, lactitol, superfloc c577, and dextran sulfate) on the stability of glutathione transferase 1 enzyme from Zea mays (ZmGSTF1-1) under free and tethered conditions at 4 and 25 °C. Among all additives, the best stabilizing effects were observed in the case of superfloc c577 and sucrose at both tested temperatures, yet at distinct concentrations at each condition. Those two stabilizing agents were further combined and potential positive synergistic effects were investigated. In addition, we assessed the long-term storage and operational stability of ZmGSTF1-1 under tethered conditions in the presence of additives, which provided the most conducive effects on its stability under free conditions. Our results strongly suggest that the presence of additives may be beneficial to the stability of the enzyme under both free and tethered conditions. Thermodynamic analysis of the free enzyme in the presence of sucrose, which exhibited the best stabilizing effect at both temperatures, shed light on the possible mechanism of action. Given the considerable importance of the development of GST-based biosensors with prolonged stability, the present work may be of general interest to researchers in the field of applied enzymology., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.

The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is L-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human L-asparaginase 3 (hASNase3), which possesses L-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human L-asparaginase, termed hASNase1. This bacterial-type cytoplasmic L-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the L-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.

We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase., (Copyright © 2013 Elsevier Inc. All rights reserved.)

l-asparaginases hydrolyze l-asparagine to l-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses l-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits α and β) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichiacoli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the β-subunit complexed to the α-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the β-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of l-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 °C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 10 5 droplets per second, an analytical throughput larger than those obtained using flow cytometry., (Copyright © 2013 AIP Publishing LLC.)

Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form and undergoes self-cleavage to the active form at a very slow rate. Here, we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated posttranslational processing and explain why no other amino acid can substitute for glycine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)