From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching

Abstract

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...

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

“…[18] Aldolase activity was spectrophotometrically measured by the retro-aldol reaction using fructose-1,6-bisphosphate (FBP) as substrate. [43] The aldolase activity assays were run at room temperature following the decrease of absorbance at 340 nm (ε NADH = 6220 M -1 ·cm -1 ) for 5 minutes in 1 mL reaction mixture containing Tris-HCl buffer (40 mM, pH 8.0), NADH (0.2 μmol), α-GDH/TIM (2 U), 1.0 μmol of FBP and FBPA or the bifunctional DLF.…”

“…[17] This enzyme has the highest catalytic efficiency for DHA phosphorylation described in the literature. [18] The multi-enzyme system was completed with the regeneration of ATP catalysed by acetate kinase (Scheme 1). This multi-enzyme system is attractive since it is a one-pot/one-step route to the phosphorylated aldol adduct and we have showed its utility with the three synthetically useful DHAP-dependent aldolases and with a great variety of commercially available aldehydes.…”

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate, D‐Fructose 6‐Phosphate and Analogues

“…[15] Nevertheless, the regeneration of ATP based on acetate kinase, was changed and replaced for the phosphoenolpyruvate (PEP)/pyruvate kinase (PK) system. This latter system displays two interesting advantages for large scale biosynthesis of DHAP: (i) PEP is a more stable phosphate donor than the originally used acetylphosphate, and (ii) PK is a cheaper enzyme than acetate kinase.…”

“…FSA acceptor substrate D-glyceraldehyde-3-phosphate (D-G3P) was obtained in situ by isomerisation of DHAP using triosephosphate isomerase (TPI). Interestingly, DHAP was readily synthesised from DHA by direct phosphorylation catalysed by dihydroxyacetone kinase [15] (DHAK) with ATP regenerated in situ by the phosphoenolpyruvate (PEP)/pyruvate kinase (PK) system. The second strategy (pathway B, Scheme 1) differed from the first one by means of D-G3P formation and corresponded to a one-pot one-step reaction.…”

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

“…Recent work centered on the evolution of metalloenzymes has identified the many different ways metal-dependent activity can be tuned along evolutionary trajectories. Alterations from the "native" metal of a metalloenzyme is consistently seen to push promiscuous activity to be more prominent (71,72). Beyond direct metal preference changes, work by BenDavid and coworkers on paraoxonase-1 clearly showed how an Table S2 in the supplemental material.…”