Preparation and Characterization of a Bifunctional Aldolase/Kinase Enzyme: A More Efficient Biocatalyst for CC Bond Formation

Abstract

Abstract:A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channelling.

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…that possesses both aldolase and kinase activities. [98,99] The fusion protein consists of a monomeric fructose-1,6-diphosphate aldolase from Staphylococcus carnosus and a homodimeric dihydroxyacetone kinase from C. freundii linked by five amino acids. With this approach they accomplished a 20-fold increase in the reaction rate which they attribute to substrate channeling effects.…”

Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase

“…They are among the most important biocatalysts for C-C bond formation. 73,[120][121][122][123][124] Aldol reactions catalysed by Dha-P dependent aldolases consist in the formation of two new stereocenters with a stereochemistry controlled by the enzymes. Therefore, from two given substrates it is possible to obtain the four diastereoisomers using four different aldolases which is a powerful synthetic advantage, as shown in Scheme 4.10.…”

“…However, their major disadvantage is precisely their strict specificity for the expensive and unstable Dha-P. 73,124,126,127 This molecule is overpriced to be used in large-scale synthesis and is labile at neutral and basic pH values causing the decrease of its effective concentration with time in the enzymatic reaction media. 123 Therefore, an efficient method of Dha-P preparation is still essential. 73,128 Following this requirement, the group of Prof. Eduardo García-Junceda of the Institute of the General Organic Chemistry in Madrid have elaborated a straightforward multi-enzyme system for one-pot C-C bond formation catalysed by DHAP-dependent aldolases, based in the use of the ATP-dependent DHAK from C. freundii for in situ Dha-P formation.…”

“…73,128 Following this requirement, the group of Prof. Eduardo García-Junceda of the Institute of the General Organic Chemistry in Madrid have elaborated a straightforward multi-enzyme system for one-pot C-C bond formation catalysed by DHAP-dependent aldolases, based in the use of the ATP-dependent DHAK from C. freundii for in situ Dha-P formation. 73,120,122,123 In this system the ATP is regenerated by catalysis of acetate kinase (AK). The fusion protein preserve both kinase and aldolase activity with a very high catalytic efficiency.…”

“…The fusion protein preserve both kinase and aldolase activity with a very high catalytic efficiency. 73,120,122,123 This system is presented in the Scheme 4.11. Scheme 4.11.…”