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Title: Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NIGMS
OSTI Identifier:
1340725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 55; Journal Issue: 30
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Gagné, Donald, Narayanan, Chitra, Nguyen-Thi, Nhung, Roux, Louise D., Bernard, David N., Brunzelle, Joseph S., Couture, Jean-François, Agarwal, Pratul K., and Doucet, Nicolas. Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans. United States: N. p., 2016. Web. doi:10.1021/acs.biochem.6b00130.
Gagné, Donald, Narayanan, Chitra, Nguyen-Thi, Nhung, Roux, Louise D., Bernard, David N., Brunzelle, Joseph S., Couture, Jean-François, Agarwal, Pratul K., & Doucet, Nicolas. Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans. United States. doi:10.1021/acs.biochem.6b00130.
Gagné, Donald, Narayanan, Chitra, Nguyen-Thi, Nhung, Roux, Louise D., Bernard, David N., Brunzelle, Joseph S., Couture, Jean-François, Agarwal, Pratul K., and Doucet, Nicolas. 2016. "Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans". United States. doi:10.1021/acs.biochem.6b00130.
@article{osti_1340725,
title = {Ligand Binding Enhances Millisecond Conformational Exchange in Xylanase B2 from Streptomyces lividans},
author = {Gagné, Donald and Narayanan, Chitra and Nguyen-Thi, Nhung and Roux, Louise D. and Bernard, David N. and Brunzelle, Joseph S. and Couture, Jean-François and Agarwal, Pratul K. and Doucet, Nicolas},
abstractNote = {},
doi = {10.1021/acs.biochem.6b00130},
journal = {Biochemistry},
number = 30,
volume = 55,
place = {United States},
year = 2016,
month = 8
}
  • Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of {lambda}gtWES.{lambda}B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the levelmore » produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. Fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T.fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.« less
  • Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residuesmore » 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. As a result, such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs.« less
  • The authors have been studying the mechanism of cellulose degradation by the cellulaces of the actinomycete Thermomonospora fusca, a thermophylic, filamentous soil bacterium. Six structurally and functionally distinct cellulases, E1 to E6 have been identified and purified. In the present study the E1 and E4 genes have been sequenced and introduced into both Streptomyces lividans and Escherichia coli. The products of digestion of cellutriose, cellotetraose, cellopentaose, and swollen cellulose revealed quite different product profiles and specificities for E1 and E4. 41 refs., 8 figs., 4 tabs.
  • Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B atmore » high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.« less