Limits...
Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization.

Wang YL, Chow SY, Lin YT, Hsieh YC, Lee GC, Liaw SH - Acta Crystallogr. D Biol. Crystallogr. (2014)

Bottom Line: Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold.The N253A structure showed a small pore created for water entry.Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan.

ABSTRACT
Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8 barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the -1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

Show MeSH
Ribbon views of the DrTS dimer (a) and monomer (b) structures. The protein consists of a (β/α)8 barrel (red), subdomain B (blue), domain C (cyan) and two TS-unique modules (S7 in green and S8 in yellow). The bound Mg2+ and Ca2+ ions are shown as green and magenta spheres, respectively, with the Tris molecule shown as a stick representation. The dimeric interfaces are formed mainly by domain C and S8 and have a total buried area of ∼3000 Å2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257617&req=5

fig2: Ribbon views of the DrTS dimer (a) and monomer (b) structures. The protein consists of a (β/α)8 barrel (red), subdomain B (blue), domain C (cyan) and two TS-unique modules (S7 in green and S8 in yellow). The bound Mg2+ and Ca2+ ions are shown as green and magenta spheres, respectively, with the Tris molecule shown as a stick representation. The dimeric interfaces are formed mainly by domain C and S8 and have a total buried area of ∼3000 Å2.

Mentions: Analytical ultracentrifugation experiments were able to clearly demonstrate that DrTS exists as a dimer in solution as well as in the crystal form, with each asymmetric unit containing two and four DrTS dimers in space groups P212121 and P21, respectively (Figs. 1 ▶ and 2 ▶a). Clear electron density was observed covering the entire DrTS molecule, except for residues 1–5 and the vector linkers. The four or eight subunits in each asymmetric unit did not display significant differences, with root-mean-square deviations of 0.3–0.4 Å for all Cα atoms. As in other GH13 members, each DrTS protomer mainly consists of a catalytic (β/α)8 barrel (β1–β8, α1–α8 and α6′) as the central core and a C-terminal β-sandwich (residues 463–552; domain C) (Fig. 2 ▶b). Domain C is made up of two antiparallel β-sheets with five (Cβ1–Cβ3, Cβ5 and Cβ7) and two (Cβ4 and Cβ6) β-strands. This region is tightly connected to the (β/α)8 barrel through an extensive network of inter­actions consisting of nine hydrogen bonds and various hydrophobic patches involving 47 residues, which are mainly contributed by its five-stranded β-sheet and α6, α6′, α7 and α8. In TSs, domain C has the greatest diverse in sequence; while DrTS shares ∼50% sequence identity with MtTS overall, they display only ∼25% sequence identity in domain C.


Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization.

Wang YL, Chow SY, Lin YT, Hsieh YC, Lee GC, Liaw SH - Acta Crystallogr. D Biol. Crystallogr. (2014)

Ribbon views of the DrTS dimer (a) and monomer (b) structures. The protein consists of a (β/α)8 barrel (red), subdomain B (blue), domain C (cyan) and two TS-unique modules (S7 in green and S8 in yellow). The bound Mg2+ and Ca2+ ions are shown as green and magenta spheres, respectively, with the Tris molecule shown as a stick representation. The dimeric interfaces are formed mainly by domain C and S8 and have a total buried area of ∼3000 Å2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257617&req=5

fig2: Ribbon views of the DrTS dimer (a) and monomer (b) structures. The protein consists of a (β/α)8 barrel (red), subdomain B (blue), domain C (cyan) and two TS-unique modules (S7 in green and S8 in yellow). The bound Mg2+ and Ca2+ ions are shown as green and magenta spheres, respectively, with the Tris molecule shown as a stick representation. The dimeric interfaces are formed mainly by domain C and S8 and have a total buried area of ∼3000 Å2.
Mentions: Analytical ultracentrifugation experiments were able to clearly demonstrate that DrTS exists as a dimer in solution as well as in the crystal form, with each asymmetric unit containing two and four DrTS dimers in space groups P212121 and P21, respectively (Figs. 1 ▶ and 2 ▶a). Clear electron density was observed covering the entire DrTS molecule, except for residues 1–5 and the vector linkers. The four or eight subunits in each asymmetric unit did not display significant differences, with root-mean-square deviations of 0.3–0.4 Å for all Cα atoms. As in other GH13 members, each DrTS protomer mainly consists of a catalytic (β/α)8 barrel (β1–β8, α1–α8 and α6′) as the central core and a C-terminal β-sandwich (residues 463–552; domain C) (Fig. 2 ▶b). Domain C is made up of two antiparallel β-sheets with five (Cβ1–Cβ3, Cβ5 and Cβ7) and two (Cβ4 and Cβ6) β-strands. This region is tightly connected to the (β/α)8 barrel through an extensive network of inter­actions consisting of nine hydrogen bonds and various hydrophobic patches involving 47 residues, which are mainly contributed by its five-stranded β-sheet and α6, α6′, α7 and α8. In TSs, domain C has the greatest diverse in sequence; while DrTS shares ∼50% sequence identity with MtTS overall, they display only ∼25% sequence identity in domain C.

Bottom Line: Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold.The N253A structure showed a small pore created for water entry.Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan.

ABSTRACT
Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8 barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the -1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

Show MeSH