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.

Figure 1

¹H NMR spectroscopic product analysis of the reaction of Cg10062 with 2, 3, 8, 9, or 10. (A) Products of Cg10062 with 8 (~95 mM) after 3 min. (B) Products of Cg10062 with 2 (~63 mM) after 48 min. The signals for the unreacted 2 are not shown. (C) Products of Cg10062 with 3 (~63 mM) after 48 min. The signals for the unreacted 3 are not shown. The small amount of 5 present is consistent with nonenzymatic decarboxylation of 4. (D) Products of Cg10062 with 9 (~80 mM) after 3 min. The small amount of 12 present is consistent with the nonenzymatic decarboxylation of 11. (E) Products of Cg10062 with 10 (~80 mM) after 39 min. The amount of 12 present (2.5%) is more than expected for nonenzymatic decarboxylation. All reactions contained ~12.3 μM Cg10062 and 30 μL of DMSO-d₆. The reactions are individually scaled to show the products of the reactions. The scale can be assessed by the height of the DMSO peak (labeled in the spectra). The hydrates of 4 and 5 are also present: the signal at ~2.3 ppm corresponds to the methyl group of the hydrate of 4, and the signal at ~1.1 ppm corresponds to the methyl group of the hydrate of 5.

Figure 2

¹H NMR spectroscopic product analysis of the reaction of E114Q-Cg10062 with 2, 3, 8, 9, or 10. (A) Products of E114Q-Cg10062 with 8 (~95 mM) after 18 min. The small amount of 5 present is consistent with nonenzymatic decarboxylation of 4. The reaction is not complete with a significant amount of 8 present (identified by the singlet at 2.90 ppm). (B) Products of E114Q-Cg10062 with 2 (~63 mM) after 18 min. The small amount of 5 present is consistent with nonenzymatic decarboxylation of 4. The reaction is not complete with a significant amount of 2 still present (NMR signals not shown). (C) Products of E114Q-Cg10062 with 3 (~63 mM) after 90 min. There are no observable signals for 4 or 5. (D) Products of E114Q-Cg10062 with 9 (~80 mM) after 12 min. The signal at 2.03 ppm corresponds to the methyl group of 12. The signals at 2.08 and 3.23 ppm correspond to the methyl and methylene groups respectively of 11. (E) Products of E114Q-Cg10062 with 10 (~80 mM) after 90 min. All reactions contained ~12.3 μM E114Q-Cg10062 and 30 μL of DMSO-d₆. The reactions are individually scaled to show the product reaction. The scale can be assessed by the height of the DMSO peak, which is labeled in the spectra.

Figure 3

¹H NMR spectroscopic product analysis of the reaction of E114D-Cg10062 with 8, 9, or 10. (A) Products of E114D-Cg10062 with 8 (~95 mM) after 3 min. (B) Products of E114D-Cg10062 with 9 (~80 mM) after 3 min. (C) Products of E114D-Cg10062 with 10 (~80 mM) after 3 min. All reactions contained ~10.2 μM E114D-Cg10062 and 30 μL of DMSO-d₆. The reactions are individually scaled to show the product reaction. The scale can be assessed by the height of the DMSO peak, which is labeled in the spectra.

Figure 4

¹H NMR spectroscopic product analysis of the reaction of Y103F-Cg10062 with 2, 3, 8, 9, or 10. (A) Products of Y103F-Cg10062 with 8 (~95 mM) after 27 min. The reaction is not complete with 8 still present (~2.9 ppm). (B) Products of Y103F-Cg10062 with 2 (~63 mM) after 18 min. The reaction is not complete with 2 still present (NMR signals not shown). (C) The reaction of Y103F-Cg10062 with 3 (~63 mM) after 18 min. There are no observable signals for products except for a trace of 4 in the baseline. (D) Products of Y103F-Cg10062 with 9 (~80 mM) after 3 min. The small amount of 12 present is consistent with nonenzymatic decarboxylation of 11. (E) Products of Y103F-Cg10062 with 10 (~80 mM) after 6 min. All reactions contained ~19.1 μM Y103F Cg10062 and 30 μL of DMSO-d₆. The reactions are individually scaled to show the product reaction. The scale can be assessed by the height of the DMSO peak, which is labeled in the spectra.

Figure 5

ESI-MS spectra of the E114D mutant of Cg10062 incubated with (A) 9 and (B) 10 in the presence of NaCNBH₃. The signals at 17 085 Da and 17 084 Da (in A and B, respectively) correspond to the unlabeled E114D-Cg10062. The signals at 17 126 Da correspond to E114D-Cg10062 covalently modified by the reduced imine of 12. The smaller signals at 17 166 Da and 17 168 Da (in A and B, respectively) correspond to E114D-Cg10062 covalently modified by the reduced imine of 11.

Figure 6

ESI-MS spectra of the Y103F mutant of Cg10062 incubated with 10 in the presence of NaCNBH₃. The signal at 17 083 Da corresponds to the unlabeled Y103F-Cg10062. The signals at 17 125 Da and 17 165 Da correspond to Y103F-Cg10062 covalently modified by the reduced imine of 12 and 11, respectively.

Figure 7

MALDI-MS of spectra of fragments from the proteolytic digest of the E114D mutant of Cg10062 incubated with (A) 9 and (B) 10 and treated with NaCNBH₃. The signals for the six major peptide fragments are labeled and are assigned as follows: 985.4 Da, Leu-81 to Glu-88; 1422.2 Da, Tyr-115 to Glu-126; 1768.3 Da, Asn-61 to Glu-75; 1896.2/1896.4 Da, Pro-1 to Glu-15; 1938.3 Da, Pro-1 to Glu-15 (+ 42 Da); and 2238.2/ 2238.4 Da, Ile-107 to Glu-126. The mass difference of 42 Da on the Pro-1 to Glu-15 fragment is consistent with the covalent modification of the fragment by the reduced imine of 12. Only the Pro-1 to Glu-15 fragment has a covalent adduct.

Figure 8

MALDI-MS of spectra of fragments from the proteolytic digest of the Y103F mutant of Cg10062 incubated with 10 and treated with NaCNBH₃. The signals for the five major peptide fragments are labeled and are assigned as follows: 985.4 Da, Leu-81 to Glu-88; 1422.4 Da, Tyr-115 to Glu-126; 1768.5 Da, Asn-61 to Glu-75; 1896.4 Da, Pro-1 to Glu-15; and 1938.5 Da, Pro-1 to Glu-15 (+42 Da). The mass difference of 42 Da on the Pro-1 to Glu-15 fragment is consistent with the covalent modification of the fragment by the reduced imine of 12. Only the Pro-1 to Glu-15 fragment has a covalent adduct.

Figure 9

Bar graph showing the activities for wild type Cg10062 and the E114Q-, E114D-, and Y103F mutants of Cg10062 with 2, 8, 9, and 10. The activities for the four substrates (2, 8, 9, and 10) with Cg10062, and with the E114Q-, E114D-, and Y103F mutants of Cg10062 are shown in the green, blue, yellow, and red bars, respectively. The set of bars in the forefront (lighter shades of these colors) represents enzyme and substrate combinations showing hydratase/decarboxylase activity. The set of bars in the background (darker shades) represents enzyme and substrate combinations showing only hydratase activity. The height of each bar indicates the log (k_cat/K_m) values (in M⁻¹ s⁻¹) for the four substrates.

Scheme 1

Enzyme-Catalyzed Reactions Comprising the 1,3-Dichloropropene Catabolic Pathway

Scheme 2

(A) Enzyme-Catalyzed Conversion of 2-Oxo-3- pentynoate (6) to Acetopyruvate (7) and (B) Acetylene and Allene Substrates Used in This Work

Scheme 3

Cg10062-Catalyzed Conversion of Propiolate (8) to a Mixture of Malonate Semialdehyde (4) and Acetaldehyde (5)

Scheme 4

Cg10062-Catalyzed Conversion of 2-Butynoate (10) to a Mixture of Acetoacetate (11) and Acetone (12)

Scheme 5

Irreversible Inactivation of cis-CaaD by (R)- Oxirane-2-carboxylate (13)

Scheme 6

Catalytic Mechanism for cis-CaaD

Scheme 7

Cg10062-Catalyzed Hydration of 8 by Direct Attack of Water Followed by Decarboxylation

Scheme 8

Cg10062-Catalyzed Hydration of 8 by Direct Attack of Water Followed by Decarboxylation via a Schiff Base

Scheme 9

Cg10062-Catalyzed Hydration of 8 by a Covalent Catalysis Mechanism