In Vitro Scanning-Saturation Mutagenesis

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

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Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

Applied and Environmental Microbiology ◽

10.1128/aem.01122-12 ◽

2012 ◽

Vol 78 (18) ◽

pp. 6647-6655 ◽

Cited By ~ 29

Author(s):

Yu Zhang ◽

Jiao An ◽

Wei Ye ◽

Guangyu Yang ◽

Zhi-Gang Qian ◽

...

Keyword(s):

Catalytic Efficiency ◽

Dynamics Simulation ◽

Saturation Mutagenesis ◽

Divergent Evolution ◽

Laboratory Evolution ◽

Content Type ◽

Hydrolytic Activities ◽

Geobacillus Kaustophilus ◽

Amidohydrolase Superfamily

ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

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Saturation mutagenesis defines novel mouse models of severe spine deformity

Disease Models & Mechanisms ◽

10.1242/dmm.048901 ◽

2021 ◽

Author(s):

Jonathan J Rios ◽

Kristin Denton ◽

Hao Yu ◽

Kandamurugu Manickam ◽

Shannon Garner ◽

...

Keyword(s):

Mouse Models ◽

Human Disease ◽

Spinal Column ◽

Saturation Mutagenesis ◽

Recessive Mutation ◽

Spine Deformity ◽

Skeletal Disease ◽

Genetic Mouse Models

Embryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular, and nervous systems, which may result in deformities such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1,275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1,275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated ENU alleles in 21% of pedigrees. Our study provides proof-of-concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural, and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced trans-activation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.

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The Plant NF-Y DNA Matrix In Vitro and In Vivo

Plants ◽

10.3390/plants8100406 ◽

2019 ◽

Vol 8 (10) ◽

pp. 406 ◽

Cited By ~ 2

Author(s):

Nerina Gnesutta ◽

Matteo Chiara ◽

Andrea Bernardini ◽

Matteo Balestra ◽

David S. Horner ◽

...

Keyword(s):

Saturation Mutagenesis ◽

Sequence Specificity ◽

Histone Fold ◽

Nuclear Factor Y ◽

Histone Fold Domain ◽

Genes Encoding ◽

Core Histones ◽

Ccaat Box

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

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Development of a Single-Gene, Signature-Tag-Based Approach in Combination with Alanine Mutagenesis To Identify Listeriolysin O Residues Critical for theIn VivoSurvival of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.06196-11 ◽

2012 ◽

Vol 80 (6) ◽

pp. 2221-2230 ◽

Cited By ~ 13

Author(s):

Jody A. Melton-Witt ◽

Susannah L. McKay ◽

Daniel A. Portnoy

Keyword(s):

Listeria Monocytogenes ◽

Single Gene ◽

Saturation Mutagenesis ◽

Single Amino Acid ◽

Screening Methods ◽

Listeriolysin O ◽

Phenotypic Analysis ◽

Content Type

ABSTRACTListeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogenListeria monocytogenes. LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis ofL. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termedinvivoanalysis bysaturation mutagenesis andsignature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzedin vivoeach conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using commonin vitrotechniques suggested that the functional contributions of some residues could only have been revealed throughin vivoanalysis.

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Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Frontiers in Catalysis ◽

10.3389/fctls.2021.774512 ◽

2021 ◽

Vol 1 ◽

Author(s):

Eun Young Hong ◽

Sun-Gu Lee ◽

Hyungdon Yun ◽

Byung-Gee Kim

Keyword(s):

Disulfide Bond ◽

Specific Activity ◽

Catalytic Efficiency ◽

Arginine Decarboxylase ◽

Saturation Mutagenesis ◽

Alkaline Ph ◽

Wild Type ◽

Active Site Residues ◽

Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).

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Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis

10.1101/2020.03.17.996157 ◽

2020 ◽

Cited By ~ 2

Author(s):

Snigdha Sarkar ◽

Wenjia Gu ◽

Eric W. Schmidt

Keyword(s):

Enzymatic Synthesis ◽

Cyclic Peptides ◽

Chemical Space ◽

Saturation Mutagenesis ◽

Recognition Sequence ◽

Drug Candidates ◽

New Class ◽

Dual Action ◽

The Right

ABSTRACTCyclic peptides are excellent drug candidates, placing macrocyclization reactions at the apex of drug development. PatG and related dual-action proteases from cyanobactin biosynthesis are responsible for cleaving off the C-terminal recognition sequence and macrocyclizing the substrate to provide cyclic peptides. This reaction has found use in the enzymatic synthesis of diverse macrocycles. However, these enzymes function best on substrates that terminate with the non-proteinogenic thiazole/thiazoline residue, complicating synthetic strategies. Here, we biochemically characterize a new class of PatG-like macrocyclases that natively use proline, obviating the necessity of additional chemical or biochemical steps. We experimentally define the biochemical steps involved in synthesizing the widespread prenylagaramide-like natural products, including macrocyclization and prenylation. Using saturation mutagenesis, we show that macrocyclase PagG and prenyltransferase PagF are highly promiscuous, producing a library of more than 100 cyclic peptides and their prenylated derivatives in vitro. By comparing our results to known cyanobactin macrocyclase enzymes, we catalog a series of enzymes that collectively should synthesize most small macrocycles. Collectively, these data reveal that, by selecting the right cyanobactin macrocyclase, a large array of enzymatically synthesized macrocycles are accessible.

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Molecular Analysis of Dasatinib Resistance Mechanisms in CML Patients Identifies Novel BCR-ABL Mutations Predicted To Retain Sensitivity to Imatinib: Rationale for Combination Tyrosine Kinase Inhibitor Therapy.

Blood ◽

10.1182/blood.v106.11.1093.1093 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1093-1093 ◽

Cited By ~ 2

Author(s):

Neil P. Shah ◽

John M. Nicoll ◽

Susan Branford ◽

Timothy P. Hughes ◽

Ronald L. Paquette ◽

...

Keyword(s):

Phase I ◽

Kinase Inhibitor ◽

Acquired Resistance ◽

Kinase Domain ◽

Saturation Mutagenesis ◽

Imatinib Resistant ◽

T315i Mutation ◽

Resistant Mutation ◽

Phase I And Ii

Abstract Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1). Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (<5% blasts with partial recovery of peripheral blood counts) on dasatinib 90 mg orally given twice daily, but relapsed with MBC after six months. Sequence analysis of the BCR-ABL kinase domain at the time of relapse revealed the presence of the imatinib-resistant mutation M244V as well as the novel mutation T315A. This finding is of particular interest because T315A and several other novel BCR-ABL mutations were recently recovered in a saturation mutagenesis study designed to define potential mechanisms of dasatinib resistance. Remarkably, many of these mutations retain sensitivity to imatinib in vitro (Burgess et al, PNAS, 2005). Through periodic molecular monitoring of other dasatinib-treated patients, we have identified a second novel BCR-ABL mutant, F317I, that developed in an imatinib-resistant CP patient after 9 months of treatment. Similar to T315A, F317I was isolated in the saturation mutagenesis screen for dasatinib resistance and is predicted to retain sensitivity to imatinib. Taken together, our findings implicate the T315I mutation as the principle mechanism of resistance to dasatinib, but more importantly, strongly support the use for combination kinase inhibitor therapy in CML to prevent emergence of drug resistant clones. A phase I trial to assess the safety of combining imatinib with dasatinib is planned.

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