Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation

Abstract

Cg10062 is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue from Corynebacterium glutamicum with an unknown function and an uninformative genomic context. It shares 53% pairwise sequence similarity with cis-CaaD including the six active site amino acids (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity. However, Cg10062 is a poor cis-CaaD: it lacks catalytic efficiency and isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene compound, 2,3-butadienoate, were investigated as potential substrates. Cg10062 functions as a hydratase/decarboxylase using propiolate as well as the cis-3-chloro- and 3-bromoacrylates, generating mixtures of malonate semialdehyde and acetaldehyde. The two activities occur sequentially at the active site using the initial substrate. With 2,3-butadienoate and 2-butynoate, Cg10062 functions as a hydratase and converts both to acetoacetate. Mutations of the proposed water-activating residues (E114Q, E114D, and Y103F) have a range of consequences from a reduction in wild type activity to a switch of activities (i.e., hydratase into a hydratase/decarboxylase or vice versa). The intermediates for the hydration and decarboxylation products can be trapped as covalent adducts to Pro-1 when NaCNBH3 is incubated with the E114D mutant and 2,3-butadienoate or 2- butynoate, and the Y103F mutant and 2-butynoate. Three mechanisms are presented to explain these findings. One mechanism involves the direct attack of water on the substrate, whereas the other two mechanisms use covalent catalysis in which a covalent bond forms between Pro-1 and the hydration product or the substrate. The strengths and weaknesses of the mechanisms and the implications for Cg10062 function are discussed.

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides

“…Interestingly, a beauvericin derivative with incorporated propargyl-lactate could not be detected, although the hydroxy acid had been accepted in in vitro and in vivo experiments in E. coli [ 45 ]. This could be due to a metabolization of the acid by A. niger as also observed in Corynebacterium glutamicum [ 49 ]. Surprisingly, SB19.23 was not able to synthesize any artificial bassianolide derivative, suggesting that the substrate specificity of the BassSyn is tightly controlled compared to BeauvSyn.…”

Synthesis and enzymatic ketonization of the 5-(halo)-2-hydroxymuconates and 5-(halo)-2-hydroxy-2,4-pentadienoates

“…The 1 H, 13 C, and 19 F NMR data are presented in Supporting Information File 1 . The approximate percentages of the components were determined by integration and are summarized in Table 3 [ 15 ]. The highly electronegative fluoride substituent in 5d prevents the detectable formation of the corresponding α,β-unsaturated ketone, 9d .…”

“…The spectra for the final mixtures were similar to those recorded for the samples that were allowed to equilibrate in buffer overnight. The approximate amounts of product in the mixtures were determined by integration of the signals, as described previously [ 15 , 26 ]. The C3 methylene protons show signals in the range of 3.39–3.53 ppm (2.41–2.53 ppm if hydrated) and the C5 methylene protons show signals in the range of 4.07–4.16 ppm.…”

Mechanistic studies on the metal-free decarboxylative coupling reaction for synthesis of propargylamines by NMR

“…4,5 Since then, several related methods have been independently developed by us and other groups. [6][7][8][9][10][11][12][13][14][15][16] The decarboxylative coupling of alkynylcarboxylic acids showed a similar reaction pattern to the Sonogashira reaction of terminal alkynes. Therefore, alkynylcarboxylic acids have been used as the surrogates of terminal alkynes in the coupling reactions and three-component reactions.…”