Reactive Enamines and Imines In Vivo: Lessons from the RidA Paradigm

Abstract

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites.

Keywords: 2-aminoacrylate stress; RidA; enamine/imine metabolism; reactive metabolite.

Figure I

Generalized scheme showing coordination of pyridine N and 3’O from pyridoxal 5’-phosphate (black) for the four main fold types of PLP-dependent enzymes. Since fold types V-VII contain members belonging to only one reaction type, these were excluded from the scheme. Enzyme active site residues are shown in blue and the phosphate group of PLP is shown with ‘℗’. Beneath the fold type label, generators of free 2AA are highlighted in green and enzymes damaged by free 2AA are shown in red. Note, the identified residues are provided using the listed enzymes as templates; for example, fold type III decarboxylases coordinate the pyridine N with a glutamate residue replacing the listed arginine residue. A comprehensive list of PLP coordinating residues is provided by Singh et al. [86].

Figure 1,. Key Figure - Enzymatic production of free enamines and imines.

Various fold-type II PLP-dependent enzymes produce and release a reactive enamine intermediate following α,β-elimination of an amino acid precursor (A). The enamine is protonated and tautomerizes to form an iminium ion intermediate. A subset of PLP-dependent α,β-eliminases enzymatically facilitate iminium ion formation prior to its release from the active site (B). FAD- dependent enzymes can also produce an iminium ion directly (C). Equilibrium between enamine, imine, and iminium ion forms is largely dependent upon pH. In vitro, the iminium ion can react with free water, generating a stable keto acid product (D). RidA serves an important role in catalyzing the deamination of enamines and imines in vivo. Elimination of RidA leads to accumulation of free enamines, most notably 2-aminoacrylate (2AA) (R-group = H). Free 2AA can covalently modify and inactivate a number of PLP-dependent enzymes belonging to fold-type I, III, and IV (E). Enamine/imine generator and target enzymes are denoted using blue and red, respectively. Relevant coenzymes are denoted as bound circles, where PLP is colored yellow and FAD is orange.

Figure 2 –. Proposed reaction mechanism for RidA-mediated hydrolysis of 2-iminiopropionate (2IP).

A schematic for the proposed reaction mechanism of RidA using data extrapolated from the crystal structure of the RidA homolog, TdcF, with bound serine (2UYK) and 2-ketobutyrate (2UYN) [46]. The seven residues conserved among Rid family members are underlined. The substrate/product are shown in red, water is depicted in green, and RidA residues are shown in blue. The active site is at the interface of two subunits and residues from one subunit are labeled with an “A” while those from the other are labeled “B”. Darker blue colors indicate residues in the foreground and paler blue colors are used for those in the background. The carboxyl group of 2IP forms a salt bridge with the R105 side-chain, while the protonated imine is stabilized through hydrogen bonding with the backbone carbonyl groups from R105 and G31 (left). The imine is also stabilized through a cation-pi interaction with the phenyl group from Y17. The secondary amine from the C107 backbone and the carbonyl group from the E120 side chain activate water for its nucleophilic attack on C2 of the bound iminium ion. Following a rearrangement, pyruvate is produced in the active site, before it is released along with ammonium (right).

Figure 3 –. Free 2-aminoacrylate inhibits PLP-dependent enzymes by two mechanisms.

2AA is highlighted in red and enzyme active site residues are shown in blue. The phosphate group of PLP is shown with ‘℗’. (A) A PLP/pyruvate adduct is formed following addition of 2AA via a C-C linkage. (a) Tautomeric rearrangement of 2AA generates a carbanion intermediate that (b) acts as a nucleophile to attack the 4’C of PLP. (c) Hydrolysis of the imine releases ammonium and creates a PLP/pyruvate adduct covalently bound to the active site lysine residue. (d) The adduct is susceptible to base-catalyzed formation of (e) a free PLP/pyruvate adduct. (B) 2AA/PLP covalently modifies a nucleophilic active site residue. (a) 2AA forms an external aldimine with PLP. (b) The alkene from 2AA participates in the conjugated π-bond system with PLP, strengthening the electrophilic nature of Cβ and allowing nucleophilic attack by an active site residue. (c) This forms a quinonoid intermediate, which can be rearranged and protonated to produce (d) a 2AA/PLP external aldimine covalently bound to a nucleophilic active site residue.