How calcium and Bmh1 activate yeast neutral trehalase Nth1?

Miroslava Kopecka¹, Lenka Rezabkova¹, Eva Macakova¹, Petr Man², Veronika Obsilova¹

¹Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220, Prague 4, Czech Republic

Yeast neutral trehalase (Nth1, EC 3.2.1.28) is a highly conserved enzyme which was found in many organisms. Nth1 from the Saccharomyces cerevisiae hydrolyses the cytosolic disaccharide trehalose (1-α-D-glucopyranosyl α-D-glucopyranoside) into two molecules of glucose. Trehalose serves as a carbon and energy source as well as a universal stress protectant against adverse conditions like dehydration, heat or oxidation. The activity of Nth1 is regulated by PKA protein phosphorylation, yeast 14-3-3 (Bmh) protein binding and by calcium [1,2]. Specific EF-like motif D¹¹⁴TDKNYQITIED¹²⁵ is located in the N-terminus of Nth1. This motif is conserved in many Ca²⁺-binding proteins. Residues D¹¹⁴ and D¹²⁵ are probably responsible for Ca²⁺-binding and I121 is important for a correct conformation of the motif [3]. Therefore we prepared four Nth1 mutants with one-point mutation in this motif. For our study we used analytical ultracentrifugation, enzyme-kinetic measurements and hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) to reveal how the Ca²⁺ and Bmh1-binding affect the activity of Nth1. Our kinetic measurements revealed that Nth1 mutants D¹¹⁴ and D¹²⁵ are inactive and that the Bmh1-dependent activation of Nth1 is significantly more potent than the Ca²⁺-dependent one. From the HDX-MS measurements we suggest that regions surrounding the buried active site of pNth1 directly interact with Bmh1. These regions undergo a structural change and thus enable easier substrate and products entry and departure. The Ca²⁺-dependent structural changes of Nth1 revealed that region containing putative Ca²⁺-binding site and segments from the vicinity undergo a significant structural change in the presence of calcium. This might reflect an interaction between the catalytic domain and upstream located Ca²⁺-binding domain and suggest an explanation for the Ca²⁺-dependent activation of Nth1. According to HDX-MS data another three point mutations were designed to test the effect on the Nth1 activity. Sedimentation velocity measurement was used to check the oligomeric status of the Nth1 mutants.

Supported by the Grant P207/11/0455 of the Grant Agency of the Czech Republic.

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