Transaldolase from Thermoplasma acidophilum: new reactivities and applications in biocatalysi

by Massimo Di Mascio
Doctoral thesis
Date of Examination:2025-01-27
Date of issue:2025-06-27
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ricardo A. Mata
Referee:Dr. Sonja Lorenz
Sponsor:This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 956631
Persistent Address: http://dx.doi.org/10.53846/goediss-11292

 

 

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Abstract

English

In this PhD thesis, the enzyme transaldolase from the thermophilic organism Thermoplasma acidophilum (TacTAL) was studied from two related perspectives: its reactivity toward 5-carbon phosphosugars and its potential applications in biocatalysis. The non-oxidative branch of the pentose phosphate pathway maintains a delicate equilibrium, involving enzymatic reactions among sugars of various lengths. TacTAL catalyzes the transfer of a dihydroxyacetone (DHA) unit from donor substrates such as fructose-6-phosphate (F6P) or sedoheptulose-7-phosphate (S7P) (6 or 7 carbon atoms) to acceptor substrates like erythrose-4 phosphate (E4P) or glyceraldehyde-3-phosphate (G3P) (3 or 4 carbon atoms). To preserve this equilibrium, it is essential that TacTAL avoids reacting with other similar compounds present in the cell. The enzyme’s reactivity toward the most important 5-carbon phosphosugars involved in the non oxidative branch of the pentose phosphate pathway such as xylulose-5-phosphate (X5P), arabinose-5-phosphate (A5P), and ribose-5-phosphate (R5P), was investigated using biochemical, biophysical and analytical techniques including X-ray crystallography, HPLC, steady-state kinetics, and mass spectrometry. The results demonstrate that the phosphate-binding site of the enzyme plays a crucial role in preventing X5P from penetrating deep enough into the active site to react as a donor. A5P is not utilized as an acceptor and can only act as a donor with low efficiency. The lower efficiency was due to the absence of a terminal α-CH2-OH group, a feature present in the physiological substrates F6P and S7P. Meanwhile, R5P can function as an acceptor, producing octulose-8-phosphate, but reacts as a donor with very low efficiency because its non-ideal stereochemistry at the α-carbon hinders proper alignment in the active site. The TacTAL E60Q/F132Y variant (aldolase variant) catalyzes the reversible cleavage of F6P into DHA and G3P. With its exceptional thermostability and catalytic efficiency, TacTAL represents a promising tool for biocatalytic applications. Rational design and inspiration from previous studies on related enzymes were employed to further engineer the aldolase variant, expanding its substrate scope to include non-phosphorylated polar compounds and non-polar substrates. Using HPLC and HR-MS, the variants TacTAL E60Q/F132Y/R135E, E60Q/F132Y/R135V, and E60Q/F132Y were found capable of catalyzing aldol condensation reactions with DHA or, alternatively, hydroxyacetone as donors. Key reactions included the formation of 8- and 9-carbon sugars with acceptor substrates such as R5P, ribose, L-rhamnose, and galacturonic acid, as well as reactions involving less polar substrates like glyoxal and pyruvaldehyde. The structure of TacTAL in complex with the 9-carbon sugars derived from DHA and L-rhamnose or DHA and galacturonic acid was resolved, and the products of the most efficient reactions, particularly those with galacturonic acid, were characterized through NMR. This study provides valuable insights into the substrate specificity of TacTAL and its applications in biocatalysis.
Keywords: biocatalysis, biochemistry, aldolase, transaldolase
 
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