A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5-Carboxyvanillate in the Metabolic Pathway for Lignin Degradation

Abstract

Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO2 concentrations in real time, thereby permitting us to establish that C−C bond cleavage proceeds to give CO2 rather than HCO3 − as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO2 formation that gradually decreases to an equilibrium value as CO2 is nonenzymatically hydrated to HCO3 − . The burst of CO2 is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO2 is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C−C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed kcat value of 27 s − 1 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of ∼16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO2 or HCO3 − is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases. KEYWORDS: decarboxylase, membrane inlet mass spectrometry, reaction mechanism, cluster approach, density functional theory, quantum chemistry ■ INTRODUCTION5-Carboxyvanillate decarboxylase (LigW) catalyzes the formation of vanillate (3-methoxy-4-hydroxybenzoate) via the nonoxidative decarboxylation reaction shown in Scheme 1a. LigW is an integral component within the biochemical degradation pathway of lignin from plant biomass by bacteria. Although LigW is a member of the amidohydrolase superfamily (AHS), this enzyme is located within a subset from cog2159 that catalyzes decarboxylation reactions rather than the much more common hydrolysis of carboxylate and phosphate esters performed by other AHS members.1 Recently, we interrogated the chemical reaction mechanism of LigW using the enzymes from Sphingomonas paucimobilis and Novosphingobium aromaticivorans by high-resolution X-ray crystallography, mutation of active site residues, substrate−activity relationships, and product isotope eff ects. 2 These studies led to the conclusion that LigW catalyzes the decarboxylation of 5-carboxyvanillate (5-CV) via a reaction intermediate formed by protonation of the substrate at C5 prior to carbon−carbon bond cleavage, as shown in Scheme 1b.Experimental support for the existence of the C5-protonated intermediate was provided by the 1.0...

A Theoretical Study of the Benzoylformate Decarboxylase Reaction Mechanism

“…Although it has been demonstrated that entropy only has a small effect on the energies of the chemical step of enzyme reactions (Senn et al, 2005 , 2009 ; Hu and Zhang, 2006 ; Lonsdale et al, 2012 ; Kazemi et al, 2016 ; Mulholland, 2016 ), its contribution must be considered in the case of a release of a gas molecule, which is relevant for the CO 2 release considered in the current study. In line with previous studies (Blomberg and Siegbahn, 2012 ; Lind and Himo, 2014 ; Sheng et al, 2015 , 2017 ), the entropy gain can be estimated as the translational entropy for the gas molecule (calculated to 11.3 kcal/mol), which is added at all steps after the transition state of CO 2 formation. Finally, the energies of the transition states corresponding to Int2 → Int3a , Int4b → Int5 , and Int6 → Int7 steps were computed to be slightly lower than one or both of the connecting intermediates.…”

QM Cluster or QM/MM in Computational Enzymology: The Test Case of LigW-Decarboxylase

“…In both decarboxylation pathways, it is clear that the enzyme must generate an adjacent electron sink (such as the ketone carbonyl C4 since the formation of the new carbon–hydrogen bond) to stabilize the incipient carbanion at C5 prior to decarboxylation. This mechanism corresponds to that explored in the recent combined experimental and theoretical work (Sheng et al, 2017 ) where the membrane inlet mass spectrometry (MIMS) based assay is applied to study the LigW mechanism. The above-mentioned MIMS-based strategy (Sheng et al, 2017 ) was able to establish CO 2 and not as reaction product.…”

“…This mechanism corresponds to that explored in the recent combined experimental and theoretical work (Sheng et al, 2017 ) where the membrane inlet mass spectrometry (MIMS) based assay is applied to study the LigW mechanism. The above-mentioned MIMS-based strategy (Sheng et al, 2017 ) was able to establish CO 2 and not as reaction product. We have considered also the path for the bicarbonate release but our calculated PESs with the three models used give very high energetic barriers (see Table S1 ) that are not compatible with the enzymatic kinetics.…”

“…All the obtained PESs with the used models are depicted in Figure 3 . Those concerning the QM one will be compared with the values arising from the previous larger QM-cluster model study (Sheng et al, 2017 ). B3LYP optimized structures obtained employing the ONIOM-2 model of all the species are given in Figure 4 while that for the QM and ONIOM-1 models are given in Figures S2, S3.…”

“…Vladimirova et al ( 2016 ) due to the very small difference (one atom) between the inhibitor (5-NV) and substrate (5-CV). This choice has been already shown sufficient when structurally compared with larger QM clusters (Sheng et al, 2017 ). In the active site, (see Figure 1 ) the manganese ion is octahedrally coordinated to Glu-19, His-188, Asp-314, one water molecule w1 and the substrate.…”