Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium.

ABSTRACT: A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific amino acid residues to levansucrase catalysis, the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterized kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram-positive Bacillus subtilis and Gram-negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg370 and Asn252 were of high relevance in polymer compared with oligosaccharide synthesis.

SUBMITTER: Homann A

PROVIDER: S-EPMC2049016 | biostudies-other | 2007 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs.

Project description:Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, ?-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.

| S-EPMC3093834 | biostudies-literature

A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium.

Project description:A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His6-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.

| S-EPMC1392972 | biostudies-literature

Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis.

Project description:The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331----Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331----Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

| S-EPMC1151543 | biostudies-other

Structural studies of domain movement in active-site mutants of porphobilinogen deaminase from Bacillus megaterium.

Project description:The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 Å, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding.

| S-EPMC5683031 | biostudies-literature

Complete Genome of Bacillus megaterium Siphophage Slash.

Project description:The complete annotated genome sequence of Bacillus megaterium bacteriophage Slash is described here. Several key features related to morphogenesis, replication/recombination, DNA metabolism, and lysis are described. Slash also encodes a homolog of SleB, a germination-specific cell wall amidase.

| S-EPMC3861416 | biostudies-literature

Complete Genome of Bacillus megaterium Podophage Pookie.

Project description:Bacteriophage Pookie is a novel podophage, isolated from soil, which infects Bacillus megaterium. B. megaterium is an important host for large-scale recombinant protein production. Here, we present the complete genome of phage Pookie and describe its core features.

| S-EPMC4319501 | biostudies-literature

Complete Genome of Bacillus megaterium Podophage Pascal.

Project description:Podophage Pascal infects Bacillus megaterium, a commonly used model organism in biochemical research and an important industrial-scale protein production system. Here, we report the sequenced and annotated genome of Pascal and describe its prominent features. Bacteriophages such as Pascal may be valuable tools for research and industry.

| S-EPMC4319499 | biostudies-literature

Complete Genome of Bacillus megaterium Podophage Page.

Project description:Bacillus megaterium is a Gram-positive, spore-forming saprophytic inhabitant of diverse environments. It is a reservoir for industrial chemical production and is emerging as a model organism for studying sporulation and protein localization. Here, we introduce the complete genome of Page, a novel podophage infecting B. megaterium.

| S-EPMC3990757 | biostudies-literature

Complete Genome of Bacillus megaterium Siphophage Staley.

Project description:Siphophage Staley was isolated because of its ability to grow on Bacillus megaterium. Here we report the complete genome and annotation of phage Staley and describe core features. Among its interesting genes is one encoding an SleB germination protein.

| S-EPMC3853053 | biostudies-literature

Complete Genome of Bacillus megaterium Podophage Pony.

Project description:Bacillus megaterium podophage Pony was isolated from a soil sample collected in College Station, TX. Here, we report the sequencing and annotation of the 39,844-bp genome of phage Pony and describe the major features identified.

| S-EPMC3853050 | 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