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Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants

DOI: 10.1107/S2059798316001170 DOI Help

Authors: Lena Stellmacher (Universität Stuttgart) , Tatyana Sandalova (Karolinska Institutet) , Sarah Schneider (Universität Stuttgart) , Gunter Schneider (Karolinska Institutet) , Georg A. Sprenger (Universität Stuttgart) , Anne K. Samland (Universität Stuttgart)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 72 , PAGES 467 - 476

State: Published (Approved)
Published: April 2016
Diamond Proposal Number(s): 8492

Abstract: Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalBF178Y with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the [epsilon]-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to [beta]-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalBE96Q formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Journal Keywords: Heyns rearrangement; D-tagatose 6-phosphate; Schiff base; transaldolase; stereoselectivity.

Subject Areas: Medicine, Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography

Other Facilities: P13 at PETRA III, DESY

Added On: 15/08/2016 14:01

Discipline Tags:

Health & Wellbeing Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)