Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Kinetic and sequence-structure-function analysis of LinB enzyme variants with ?- and ?-hexachlorocyclohexane.

ABSTRACT: Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ?80 fold more activity for ?- and ?-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.

SUBMITTER: Pandey R

PROVIDER: S-EPMC4116220 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Kinetic and sequence-structure-function analysis of LinB enzyme variants with β- and δ-hexachlorocyclohexane.

Pandey Rinku R Lucent Del D Kumari Kirti K Sharma Pooja P Lal Rup R Oakeshott John G JG Pandey Gunjan G

PloS one 20140730 7

Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocycl ...[more]

PMID: 25076214

Similar Datasets

Kinetic and sequence-structure-function analysis of known LinA variants with different hexachlorocyclohexane isomers.

Project description:BACKGROUND: Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, ?, ? and ?, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for ?-, ?-, and ?-HCH and enantiospecificity for (+)- and (-)-? -HCH are also discussed. METHODOLOGY/PRINCIPAL FINDINGS: Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. ?-HCH was found to be the most preferred substrate by all LinA's, followed by the ? and then ? isomer. CONCLUSIONS/SIGNIFICANCE: The kinetic observations suggest that LinA-?1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.

| S-EPMC3174995 | biostudies-literature

Haloalkane dehalogenase LinB is responsible for beta- and delta-hexachlorocyclohexane transformation in Sphingobium indicum B90A.

Project description:Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform beta- and delta-hexachlorocyclohexane (beta- and delta-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with beta- and delta-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including beta-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade beta- and delta-HCH.

| S-EPMC1563659 | biostudies-literature

Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards β-hexachlorocyclohexane.

Project description:Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards β-hexachlorocyclohexane (β-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's β-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.

| S-EPMC4230811 | biostudies-literature

SARS-CoV-2 Beta and Delta variants trigger Fc effector function with increased cross-reactivity.

Project description:Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with improved disease outcome and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta and Delta infection trigger responses with significantly improved Fc cross-reactivity against global VOCs compared with D614G-infected or Ad26.COV2.S-vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence affects Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.

| S-EPMC8761540 | biostudies-literature

Differences in crystallization of two LinB variants from Sphingobium japonicum UT26.

Project description:Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8?Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

| S-EPMC3606575 | biostudies-literature

Error structure as a function of substrate and inhibitor concentration in enzyme kinetic experiments.

Project description:Optimal design of experiments as well as proper analysis of data are dependent on knowledge of the experimental error. A detailed analysis of the error structure of kinetic data obtained with acetylcholinesterase showed conclusively that the classical assumptions of constant absolute or constant relative error are inadequate for the dependent variable (velocity). The best mathematical models for the experimental error involved the substrate and inhibitor concentrations and reflected the rate law for the initial velocity. Data obtained with other enzymes displayed similar relationships between experimental error and the independent variables. The new empirical error functions were shown superior to previously used models when utilized in weighted non-linear-regression analysis of kinetic data. The results suggest that, in the spectrophotometric assays used in the present study, the observed experimental variance is primarily due to errors in determination of the concentrations of substrate and inhibitor and not to error in measuring the velocity.

| S-EPMC1146758 | biostudies-other

Complete Genome Sequence of a γ-Hexachlorocyclohexane Degrader, Sphingobium sp. Strain TKS, Isolated from a γ-Hexachlorocyclohexane-Degrading Microbial Community.

Project description:Here, we report the complete genome sequence of a γ-hexachlorocyclohexane (γ-HCH) degrader,Sphingobiumsp. strain TKS, which was isolated from a γ-HCH-degrading microbial community. The genome of TKS consists of two chromosomes and nine plasmids. Thelingenes for conversion of γ-HCH to β-ketoadipate are dispersed on chromosome 1 and three out of the nine plasmids.

| S-EPMC4824264 | biostudies-literature

Tilt series of SARS-CoV-2 virions, Wuhan/Alpha/Beta/Delta variants

Project description: Not available

| EMPIAR-11070 | biostudies-other

Common sequence variants affect molecular function more than rare variants?

Project description:Any two unrelated individuals differ by about 10,000 single amino acid variants (SAVs). Do these impact molecular function? Experimental answers cannot answer comprehensively, while state-of-the-art prediction methods can. We predicted the functional impacts of SAVs within human and for variants between human and other species. Several surprising results stood out. Firstly, four methods (CADD, PolyPhen-2, SIFT, and SNAP2) agreed within 10 percentage points on the percentage of rare SAVs predicted with effect. However, they differed substantially for the common SAVs: SNAP2 predicted, on average, more effect for common than for rare SAVs. Given the large ExAC data sets sampling 60,706 individuals, the differences were extremely significant (p-value < 2.2e-16). We provided evidence that SNAP2 might be closer to reality for common SAVs than the other methods, due to its different focus in development. Secondly, we predicted significantly higher fractions of SAVs with effect between healthy individuals than between species; the difference increased for more distantly related species. The same trends were maintained for subsets of only housekeeping proteins and when moving from exomes of 1,000 to 60,000 individuals. SAVs frozen at speciation might maintain protein function, while many variants within a species might bring about crucial changes, for better or worse.

| S-EPMC5431670 | biostudies-literature

3D-Fun: predicting enzyme function from structure.

Project description:The 'omics' revolution is causing a flurry of data that all needs to be annotated for it to become useful. Sequences of proteins of unknown function can be annotated with a putative function by comparing them with proteins of known function. This form of annotation is typically performed with BLAST or similar software. Structural genomics is nowadays also bringing us three dimensional structures of proteins with unknown function. We present here software that can be used when sequence comparisons fail to determine the function of a protein with known structure but unknown function. The software, called 3D-Fun, is implemented as a server that runs at several European institutes and is freely available for everybody at all these sites. The 3D-Fun servers accept protein coordinates in the standard PDB format and compare them with all known protein structures by 3D structural superposition using the 3D-Hit software. If structural hits are found with proteins with known function, these are listed together with their function and some vital comparison statistics. This is conceptually very similar in 3D to what BLAST does in 1D. Additionally, the superposition results are displayed using interactive graphics facilities. Currently, the 3D-Fun system only predicts enzyme function but an expanded version with Gene Ontology predictions will be available soon. The server can be accessed at http://3dfun.bioinfo.pl/ or at http://3dfun.cmbi.ru.nl/.

| S-EPMC2447717 | 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