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.

Enzyme promiscuity is a concept that in the last years is earning prominence in different fields of enzymology like biocatalysis, enzyme engineering or enzyme evolution. [1] Catalytic promiscuity is defined as the ability of an enzyme to catalyze more than one chemical transformation. [1b,c] Naturally occurring catalytic promiscuity provide the starting point for a Darwinian evolution of enzymes to new functions since this process must occur gradually, while maintaining organism fitness throughout. [2] Tawfik and co-workers [3] have provided experimental evidence for the plasticity and "evolvability" [4] of promiscuous functions. These authors propose a model by which a protein acquires a new function, without losing the original one, and gene duplication may follow the emergence of a new function, rather than initiate it. Besides the intriguing implications that this theory of divergent molecular evolution has for protein evolution, its application to promiscuous enzymes allows to design enzymes with new catalytic activities. [5] A special case of catalytic promiscuity is the shown by metalloenzymes, where the variety of metallic ions that can be incorporated in the active site increase the range of chemical transformations that can be catalyzed by the enzyme. Thus, there are several examples showing that protein modification via the covalent attachment of ligands that incorporate metal ions or by incorporation of the catalytic metal ion alone in a suitable site for coordination, is a strategy that allows to obtain enzymes with either modified or completely new catalytic activities. [6] In this communication we describe the promiscuous behaviour of the dihydroxyacetone (DHA) kinase from Citrobacter freundii strain CECT 4626. This ATP-dependent DHAK is able to catalyse, beside the transfer of the γ-phosphate of the ATP to DHA, the cyclization of the FAD to yield riboflavin 4',5'-cyclic phosphate (4',5'-cFMN) (Scheme 1). This catalytic promiscuity is modulated by the divalent cation that forms the complex with the phosphorylated substrate. Although DHAK's are widely distributed in the three biological kingdoms, only their role in the catabolism of glycerol and in methanol assimilation in microorganisms have been well characterized. [7] In the bacteria C. freundii strain DSM 30040 [8] the entire dha regulon has been cloned and characterized at molecular level. [9] The kinetic properties and mechanism of the corresponding DHAK have been described [10] and the X-ray structure of the full-length DHAK in complex with its substrates has been elucidated. [11] This kinase is the only one known with an all-α nucleotide-binding domain. [12] From a biocatalytic point of view, ATP-dependent DHAK's have been given considerable attention because their feasibility for the simple and efficient obtaining of DHAP. [13] We have reported a multi-enzyme system for one-pot C-C bond formation catalysed by DHAP-dependent aldolases, based in the use of the recombinant DHAK from C. freundii CECT 4626, for in situ DHAP formation. [14...

A new bifunctional enzyme that displays both aldolase and kinase activities has been designed and successfully used in the synthesis of aldol adducts, employing DHA as initial donor, with an increase in the reaction rate of 20-fold over the parent enzymes, which can be interpreted in terms of substrate channelling.

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

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