Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Converting Pasteurella multocida?2-3-sialyltransferase 1 (PmST1) to a regioselective ?2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

ABSTRACT: A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocida?2-3-sialyltransferase 1 (PmST1) for ?2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective ?2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1. It retained the donor substrate promiscuity of the wild-type enzyme and was used in an efficient one-pot multienzyme (OPME) system to selectively catalyze the sialylation of the terminal galactose residue in a multigalactose-containing tetrasaccharide lacto-N-neotetraoside.

SUBMITTER: McArthur JB

PROVIDER: S-EPMC5333693 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Converting Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) to a regioselective α2-6-sialyltransferase by saturation mutagenesis and regioselective screening.

McArthur John B JB Yu Hai H Zeng Jie J Chen Xi X

Organic & biomolecular chemistry 20170130 7

A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activ ...[more]

PMID: 28134951

Similar Datasets

PmST2: a novel Pasteurella multocida glycolipid α2-3-sialyltransferase.

Project description:Pasteurella multocida (Pm) is a multi-species pathogen that causes diseases in animals and humans. Sialyltransferase activity has been detected in multiple Pm strains and sialylation has been shown to be important for the pathogenesis of Pm. Three putative sialyltransferase genes have been identified in Pm genomic strain Pm70. We have reported previously that a Pm0188 gene homolog in Pm strain P-1059 (ATCC 15742) encodes a multifunctional sialyltransferase (PmST1). We demonstrate here that while PmST1 prefers to use oligosaccharides as acceptors, PmST2 encoded by the Pm0508 gene homolog in the same Pm strain is a novel glycolipid α2-3-sialyltransferase that prefers to use lactosyl lipids as acceptor substrates. PmST2 and PmST1 thus complement each other for an efficient synthesis of α2-3-linked sialosides with or without lipid portion. In addition, β1-4-linked galactosyl lipids are better PmST2 substrates than β1-3-linked galactosyl lipids. PmST2 has been used successfully in the preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3), which is an important glycolipid and an intermediate for synthesizing more complex glycolipids such as gangliosides.

| S-EPMC3150114 | biostudies-literature

Engineering allosteric inhibition of homoserine dehydrogenase by semi-rational saturation mutagenesis screening.

Project description:Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

| S-EPMC10791936 | biostudies-literature

α2-6-Neosialidase: A Sialyltransferase Mutant as a Sialyl Linkage-Specific Sialidase.

Project description:The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

| S-EPMC5978697 | biostudies-literature

Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based Automated Screening for Protein Engineering.

Project description:We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-of-function mutants were not significantly different from the expected values for the primers among the three different suppliers. However, deep sequencing analysis revealed that the ratios of nucleotides in the primers were highly biased among the suppliers. Biases for NNB were less severe than for NNN. We applied this method to screen a fusion protein of two chitinases, ChiA and ChiB (ChiAB). Three NNB codons as well as tyrosine and serine (X1YSX2X3) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X3 position in water-soluble, active ChiAB-X1YSX2X3 mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X3 residue plays an important role in structure stabilization.

| S-EPMC6130897 | biostudies-literature

Saturation mutagenesis of disease-associated regulatory elements

Project description:The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we performed saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitution and deletion mutations. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or various integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and also comprise a gold-standard dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

2019-02-15 | GSE126550 | GEO

Plasmid-based one-pot saturation mutagenesis.

Project description:Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.

| S-EPMC5666567 | biostudies-literature

Saturation mutagenesis reveals manifold determinants of exon definition

Project description:To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts we conducted saturation mutagenesis of a 51 nt internal exon in a 3-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high throughput genetics, 5560 minigene molecules were assayed for splicing in HEK293 cells. Over 70% of mutations produced substantial (>2X) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements only a single 15 nt stem-loop, showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1Aa, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51 nt test exon simultaneously. Surprisingly, such correlations extended to weak relative protein-sequence affinities. These myriad protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.

2017-11-27 | GSE105785 | GEO

Site saturation mutagenesis of 500 human protein domains

Project description:Missense variants that change the amino acid sequences of proteins cause one third of human genetic diseases. Tens of millions of missense variants exist in the current human population, with the vast majority having unknown functional consequences. Here we present the first large-scale experimental analysis of human missense variants. Using DNA synthesis and cellular selection experiments we quantify the impact of >500,000 variants on the abundance of >500 human protein domains. This dataset, Domainome 1.0, reveals that >60% of disease-causing variants destabilize proteins. The contribution of stability to protein fitness varies across proteins and diseases, and is particularly important in recessive disorders. Combining experimental stability measurements with large language models we annotate functionally important sites across domains. Fitting energy models to the data demonstrates the conservation of mutation effects in homologous domains and allows stability to be accurately predicted for entire domain families. Domainome 1.0 demonstrates the feasibility of assaying human protein variant effects at scale and provides a large consistent reference dataset for clinical variant interpretation and the training and benchmarking of computational methods.

2024-04-27 | GSE265942 | GEO

Saturation Mutagenesis by Efficient Free-Energy Calculation.

Project description:Single-point mutations in proteins can greatly influence protein stability, binding affinity, protein function or its expression per se. Here, we present accurate and efficient predictions of the free energy of mutation of amino acids. We divided the complete mutational free energy into an uncharging step, which we approximate by a third-power fitting (TPF) approach, and an annihilation step, which we approximate using the one-step perturbation (OSP) method. As a diverse set of test systems, we computed the solvation free energy of all amino acid side chain analogues and obtained an excellent agreement with thermodynamic integration (TI) data. Moreover, we calculated mutational free energies in model tripeptides and established an efficient protocol involving a single reference state. Again, the approximate methods agreed excellently with the TI references, with a root-mean-square error of only 3.6 kJ/mol over 17 mutations. Our combined TPF+OSP approach does show not only a very good agreement but also a 2-fold higher efficiency than full blown TI calculations.

| S-EPMC5813279 | biostudies-literature

Highly efficient libraries design for saturation mutagenesis.

Project description:Saturation mutagenesis is a semi-rational approach for protein engineering where sites are saturated either entirely or partially to include amino acids of interest. We previously reported on a codon compression algorithm, where a set of minimal degenerate codons are selected according to user-defined parameters such as the target organism, type of saturation and usage levels. Here, we communicate an addition to our web tool that considers the distance between the wild-type codon and the library, depending on its purpose. These forms of restricted collections further reduce library size, lowering downstream screening efforts or, in turn, allowing more comprehensive saturation of multiple sites. The library design tool can be accessed via http://www.dynamcc.com/dynamcc_d/. Graphical Abstract.

| S-EPMC9205323 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data