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

2007 ◽  
Vol 407 (2) ◽  
pp. 189-198 ◽  
Author(s):  
Arne Homann ◽  
Rebekka Biedendieck ◽  
Sven Götze ◽  
Dieter Jahn ◽  
Jürgen Seibel
Keyword(s):  
Amino Acid ◽  
Bacillus Megaterium ◽  
Structural Information ◽  
Oligosaccharide Synthesis ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
Specific Amino Acid ◽  
Product Specificity ◽  
Structural Differences ◽  
A Cell

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.

A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 918-924 ◽  
Author(s):  
Eileen Collins Tozer ◽  
Elizabeth K. Baker ◽  
Mark H. Ginsberg ◽  
Joseph C. Loftus
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Single Amino Acid ◽  
Chimeric Receptor ◽  
Amino Acid Residues ◽  
Genetic Approach ◽  
Specific Amino Acid ◽  
Transfected Cells ◽  
Binding Function ◽  
A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 918-924 ◽  
Author(s):  
Eileen Collins Tozer ◽  
Elizabeth K. Baker ◽  
Mark H. Ginsberg ◽  
Joseph C. Loftus
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Single Amino Acid ◽  
Chimeric Receptor ◽  
Amino Acid Residues ◽  
Genetic Approach ◽  
Specific Amino Acid ◽  
Transfected Cells ◽  
Binding Function ◽  
A Cell

An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

scholarly journals Identification of Key Amino Acid Residues Determining Product Specificity of 2,3-Oxidosqualene Cyclase in Siraitia grosvenorii

Catalysts ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 577 ◽  
Author(s):  
Jing Qiao ◽  
Jiushi Liu ◽  
Jingjing Liao ◽  
Zuliang Luo ◽  
Xiaojun Ma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Bioactive Molecules ◽  
Amino Acid Residues ◽  
Product Specificity ◽  
Oxidosqualene Cyclase ◽  
Siraitia Grosvenorii ◽  
Initial Substrate ◽  
New Strategy ◽  
Key Residues

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during the initial substrate preorganization step, oxidosqualene cyclases (OSCs) are roughly segregated into a dammarenyl cation group that predominantly catalyzes triterpenoid precursor products and a protosteryl cation group which mostly generates sterol precursor products. The mechanism of conversion between two scaffolds is not well understood. Previously, we have characterized a promiscuous OSC from Siraitia grosvenorii (SgCS) that synthesizes a novel cucurbitane-type triterpene cucurbitadienol as its main product. By integration of homology modeling, molecular docking and site-directed mutagenesis, we discover that five key amino acid residues (Asp486, Cys487, Cys565, Tyr535, and His260) may be responsible for interconversions between chair–boat–chair and chair–chair–chair conformations. The discovery of euphol, dihydrolanosterol, dihydroxyeuphol and tirucallenol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into cucurbitane-type and lanostane-type skeletons, and provide a new strategy to identify key residues determining OSC specificity.

scholarly journals Genetic Diversity of the cagA gene of Helicobacter pylori strains from Sudanese Patients with Different Gastroduodenal Diseases

2019 ◽  
Author(s):  
Hadeel Gassim Hassan ◽  
Abeer Babiker Idris ◽  
Mohamed A. Hassan ◽  
Hisham N. Altayb ◽  
Kyakonye Yasin ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Amino Acid ◽  
Novel Mutation ◽  
Amino Acid Residues ◽  
Specific Amino Acid ◽  
Gastrointestinal Malignancy ◽  
High Incidence ◽  
H Pylori ◽  
High Level ◽  
Chloride Method

AbstractBackgroundThere is an increase in the prevalence of Helicobacter pylori infection in Sudan, accompanied by a high incidence of upper gastrointestinal malignancy. The cytotoxin-associated gene cagA gene is a marker of a pathogenicity island (PAI) in H. pylori and plays a crucial role in determining the clinical outcome of Helicobacter infections.ObjectiveThis study aimed to determine the frequency and heterogeneity of the cagA gene of H. pylori and correlate the presence of cagA gene with clinical outcomes.Materials and methodsFifty endoscopy biopsies were collected from Fedail and Soba hospitals in Khartoum state. DNA was extracted using the Guanidine chloride method followed by PCR to amplify 16S rRNA and cagA gene of H. pylori using specific primers. DNA amplicons of cagA gene were purified and sequenced. Bioinformatics and statistical analysis were done to characterize and to test the association between cagA gene and gastric complications.ResultsCagA gene was detected in 20/37(54%) of the samples that were found positive for H. pylori. There was no association between endoscopy finding and the presence of the cagA gene (p = 0.225). Specific amino acid variations were found at seven loci related to strains from a patient with duodenitis, gastric ulcer, and gastric atrophy (R448H, T457K, S460L, IT463-464VA, D470E, A482Q, KNV490-491-492TKT) while mutations in cancerous strain were A439P, T457P, and H500Y.ConclusionDisease-specific variations of cagA of H. pylori strains, in the region of amino acid residues 428-510, were evident among Sudanese patients with different gastroduodenal diseases. A novel mutation (K458N) was detected in a patient with duodenitis, which affects the positive electrostatic surface of cagA. Phylogenetic analysis showed a high level of diversity of cagA from Sudanese H. pylori strains.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

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