Reaction of <i>cis</i>-3-Chloroacrylic Acid Dehalogenase with an Allene Substrate, 2,3-Butadienoate: Hydration via an Enamine

Schroeder, Gottfried K.; Johnson, William H.; Huddleston, Jamison P.; Serrano, Héctor; Johnson, Kenneth A.; Whitman, Christian P.

doi:10.1021/ja206873f

Cited by 18 publications

(88 citation statements)

References 48 publications

(147 reference statements)

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“…The chemical shifts for 4, 5, their hydrates, 11, and 12 are reported elsewhere. 13,21 The approximate amount of product in the mixtures was determined by integration, as described previously. 1 …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Methodsmentioning

confidence: 99%

“…3,21 After a 48-h incubation period, the resulting peptide mixture was analyzed by MALDI- MS, as described previously. 3,21 …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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“…For these reasons, it was anticipated that Cg10062 would function as a cis-CaaD with the same mechanism and activity even though there did not appear to be a catabolic pathway for 1 in C. glutamicum (based on the genomic context). However, a comparison of the kinetic parameters for cis-CaaD and Cg10062 showed that 2 is a poor substrate for Cg10062 due to a high value of K m (72,000 vs 200 μM) resulting in an overall k cat /K m of 14 M À 1 s À 1 (1570-fold lower than that measured for cis-CaaD) (Poelarends et al, 2008a;Schroeder et al, 2012Schroeder et al, , 2013. In addition, Cg10062 processed the trans isomer, albeit poorly (0.8 M À 1 s À 1 using 3) (Poelarends et al, 2008a).…”

Section: Characterization Of Cg10062mentioning

confidence: 95%

“…cis-CaaD functions as a hydratase using 2, 11 (k cat / K m $6.4 Â 10 3 M À 1 s À 1 , as noted above), and 12 (k cat / K m $8.7 Â 10 3 M À 1 s À 1 ) (Schroeder et al, 2012). cis-CaaD processes 14 to 13, (determined by 1 H NMR spectroscopy), but the rate is too slow to obtain kinetic parameters.…”

Section: Deciphering the Reaction Mechanism Of Cg10062mentioning

confidence: 99%

The accidental assignment of function in the tautomerase superfamily

Huddleston

Johnson

Schroeder

et al. 2015

Perspectives in Science

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Cg10062 from Corynebacterium glutamicum is a tautomerase superfamily member with the characteristic β Àα À β fold and catalytic Pro-1. It is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue with high sequence similarity (53%) that includes the six critical active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114). However, Cg10062 is a poor cis-CaaD: it has much lower catalytic efficiency and lacks isomer specificity. Two acetylene compounds (propiolate and 2-butynoate) and an allene (2,3-butadienote) were investigated as potential substrates for Cg10062. Cg10062 is a hydratase/decarboxylase using propiolate and cis-3-chloro-and 3-bromoacrylates, where malonate semialdehyde is the product of hydration and acetaldehyde is the product of decarboxylation. The two activities occur consecutively using the initial substrate. In contrast, 2-butynoate and 2,3-butadienote only undergo a hydration reaction with Cg10062 to afford acetoacetate. cis-CaaD does not function as a hydratase/decarboxylase using any of these substrates, yielding only the products of hydration. Cg10062 proceeds by direct hydration or covalent catalysis (using Pro-1) depending on the substrate. Direct hydration yields the hydration products and covalent catalysis yields the hydration and decarboxylation products. Cg10062 mutants shift the reaction toward one or the other mechanism. The observation that propiolate is the best substrate suggests that Cg10062 could be a hydratase/decarboxylase in a pathway that transforms an unknown acetylene compound to acetaldehyde via propiolate. The bifunctional activity of Cg10062 might also have implications for the evolution of the dehalogenase and decarboxylase activities in the tautomerase superfamily.

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“…3). 10 As part of our ongoing research programs of allenes, 11 herein, we report a general and efficient …”

Section: Introductionmentioning

confidence: 99%

Oxone-promoted hydration of electron-deficient allenic esters and ketones into 1,3-dicarbonyl compounds

Zheng

Nie

et al. 2015

Tetrahedron Letters

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Reaction of cis-3-Chloroacrylic Acid Dehalogenase with an Allene Substrate, 2,3-Butadienoate: Hydration via an Enamine

Cited by 18 publications

References 48 publications

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

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

The accidental assignment of function in the tautomerase superfamily

Oxone-promoted hydration of electron-deficient allenic esters and ketones into 1,3-dicarbonyl compounds

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