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...