Investigation of the molecular determinants of the substrate- and stereoselectivity of dihydroxyacetone- dependent aldolases and transaldolases
Zusammenfassung der Projektergebnisse
Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond forming enzymes and are closely related, however they belong to different enzyme classes (transferases vz. lyases). To understand the differences between the two enzymes better, amino acid residues which are responsible for binding the catalytic water molecule were interchanged between FSAA and TalB. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6-phosphate aldolase and transaldolase activity, stability, pH dependence of activity, pKa value of the essential E96Q F178Y lysine residue and their three dimensional structure. The double variant TalB showed improved aldolase activity with an apparent kcat of 4.3 s^-1 . The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences which appear to be important for the discrimination between the two enzyme acitivities: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid. Furthermore, we conclude that a glutamate residue is the key player as general acid/base in the transaldolase mechanism, whereas a tyrosine residue takes over this function in fructose 6-phosphate aldolases. Attempts to obtain TalB variants with an altered stereospecificity towards 3S,4S (tagatose-6-phosphate, Tag6P, as donor) were not successful. Most likely, this is due to the irreversible inactivation of the TalB variant, TalB F178Y by Tag6P. A novel structure of a covalently bound ring sugar at the active site lysine residue of the TalB F178Y variant was revealed (in collaboration with the groups of Tatjana Sandalova and Gunter Schneider at Karolinska Institute, Stockholm, Sweden). Using kinetic inhibition studies and mass spectrometry we showed that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited irreversibly by Tag6P, whereas no inhibition was observed for wild-type TalB of E. coli. The crystal structure of the variant TalB^F178Y with bound Tag6P 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 carbon atom to the ε-NH2-group of the active site residue Lys132 and forms a five-membered ring structure. The furanose ring of the covalent adduct is formed via a so-called Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by a Tyr residue. The crystal structure of the inhibitor complex was compared to the structure of the Schiff base intermediate of TalB^E96Q 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 carbon atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the enzyme’s active site.
Projektbezogene Publikationen (Auswahl)
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(2012) Computational tools for rational protein engineering of aldolases. Comput. Struct. Biotechnol. J. 2: e201209016
Widmann M, Pleiss J, Samland AK
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(2012) Conservation of structure and mechanism within the transaldolase enzyme family. FEBS J. 279: 766-778
Samland, AK, Baier, S, Schürmann, M, Inoue, T, Huf, S, Schneider, G, Sprenger, GA, Sandalova, T
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(2012) FSAB: A new fructose-6-phosphate aldolase from Escherichia coli. Cloning, overexpression and comparative kinetic characterization with FSAA. J. Mol. Catal. B: Enzyme 84: 9-14
Sánchez-Moreno, I, Nauton, L, Théry, V, Pinet, A, Petit, J-L, de Berardinis, V, Samland, AK, Guérard-Hélaine, C, Lemaire, M
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(2015) Acid-base catalyst discriminates between a fructose 6-phosphate aldolase and a transaldolase. ChemCatChem, 7: 3140-3151
Stellmacher, L, Sandalova, T, Leptihn, S, Schneider, G, Sprenger, GA, Samland, AK
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(2015) Synthetic potential of Dihydroxyacetone-utilizing aldolases. in Industrial Biocatalysis (Ed.: P. Grunwald), Pan Stanford Publishing, Singapore, pp. 783-816
Samland, AK, Sprenger, GA
