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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.

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The interaction networks between subdomains B (magenta) and S7 (blue) in MtTS (PDB entry 4lxf; chain B) (a), MsTS (b), DrTS–Tris (c) and DrTS-N253A–Tris (d). In one MtTS structure the active site is wide open owing to a disordered region in S7, while in MsTS Leu344 blocks the substrate-binding pocket. On the other hand, in DrTS a closed conformation was observed. These TS structures indicate that subdomains B and S7 serve as a gateway for the opening and closing of the active site. In N253A, the absence of the hydrogen bond between Asn253 and Glu324 causes movement of the Glu324 side chain, leading to the creation of a small pore for water entry.
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fig8: The interaction networks between subdomains B (magenta) and S7 (blue) in MtTS (PDB entry 4lxf; chain B) (a), MsTS (b), DrTS–Tris (c) and DrTS-N253A–Tris (d). In one MtTS structure the active site is wide open owing to a disordered region in S7, while in MsTS Leu344 blocks the substrate-binding pocket. On the other hand, in DrTS a closed conformation was observed. These TS structures indicate that subdomains B and S7 serve as a gateway for the opening and closing of the active site. In N253A, the absence of the hydrogen bond between Asn253 and Glu324 causes movement of the Glu324 side chain, leading to the creation of a small pore for water entry.

Mentions: The crystal structure of MtTS showed two conformations (Roy et al., 2013 ▶). In one conformation, residues 353–381 in S7 are disordered and the active site is wide open to solvent (Figs. 7 ▶c and 8 ▶a). On the other hand, the other MtTS structure is nearly identical to the MsTS structure (Fig. 7 ▶c). S7 is composed of two short α-helices along with an extended loop, and the transition-state stabilizer Asp was shifted away from the active site by ∼4 Å (Caner et al., 2013 ▶; Roy et al., 2013 ▶). In addition, Leu344 in MsTS, which is Leu352 in MtTS, projects into the active site and blocks the substrate-binding pocket (Fig. 8 ▶b). Compared with some GH13 members (Fig. 4 ▶), the active-site residues in the MsTS and MtTS structures adopt different spatial positions, and in addition the transition-state stabilizer Asp does not interact with its surrounding residues.


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)

The interaction networks between subdomains B (magenta) and S7 (blue) in MtTS (PDB entry 4lxf; chain B) (a), MsTS (b), DrTS–Tris (c) and DrTS-N253A–Tris (d). In one MtTS structure the active site is wide open owing to a disordered region in S7, while in MsTS Leu344 blocks the substrate-binding pocket. On the other hand, in DrTS a closed conformation was observed. These TS structures indicate that subdomains B and S7 serve as a gateway for the opening and closing of the active site. In N253A, the absence of the hydrogen bond between Asn253 and Glu324 causes movement of the Glu324 side chain, leading to the creation of a small pore for water entry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: The interaction networks between subdomains B (magenta) and S7 (blue) in MtTS (PDB entry 4lxf; chain B) (a), MsTS (b), DrTS–Tris (c) and DrTS-N253A–Tris (d). In one MtTS structure the active site is wide open owing to a disordered region in S7, while in MsTS Leu344 blocks the substrate-binding pocket. On the other hand, in DrTS a closed conformation was observed. These TS structures indicate that subdomains B and S7 serve as a gateway for the opening and closing of the active site. In N253A, the absence of the hydrogen bond between Asn253 and Glu324 causes movement of the Glu324 side chain, leading to the creation of a small pore for water entry.
Mentions: The crystal structure of MtTS showed two conformations (Roy et al., 2013 ▶). In one conformation, residues 353–381 in S7 are disordered and the active site is wide open to solvent (Figs. 7 ▶c and 8 ▶a). On the other hand, the other MtTS structure is nearly identical to the MsTS structure (Fig. 7 ▶c). S7 is composed of two short α-helices along with an extended loop, and the transition-state stabilizer Asp was shifted away from the active site by ∼4 Å (Caner et al., 2013 ▶; Roy et al., 2013 ▶). In addition, Leu344 in MsTS, which is Leu352 in MtTS, projects into the active site and blocks the substrate-binding pocket (Fig. 8 ▶b). Compared with some GH13 members (Fig. 4 ▶), the active-site residues in the MsTS and MtTS structures adopt different spatial positions, and in addition the transition-state stabilizer Asp does not interact with its surrounding residues.

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
Related in: MedlinePlus