cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH4 by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The result may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.

The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-α-d-manno-heptose; modifications to the stereochemistry at C3–C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-α-d-manno-heptose has been described previously, but using enzymes from Aneurinibacillus thermoaerophilus DSM 10155. Here we describe the complete synthesis of GDP-d-glycero-α-d-manno-heptose using enzymes from C. jejuni NTCC 11168: Cj1152 and Cj1423–Cj1425. Our results yield kinetic parameters for these enzymes and outline a successful strategy for milligram–gram scale synthesis of GDP-d-glycero-α-d-manno-heptose. This achievement is critical for the characterization of other carbohydrate tailoring enzymes, which are expected to utilize GDP-d-glycero-α-d-manno-heptose for the biosynthesis of more complex carbohydrates in the CPS of C. jejuni.

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...

The bacterial degradation of the nematicide 1,3-dichloropropene, an isomeric mixture, requires the action of trans- and cis-3-chloracrylic acid dehalogenase (CaaD and cis-CaaD, respectively). Both enzymes are tautomerase superfamily members and share a core catalytic mechanism for the hydrolytic dehalogenation of the respective isomer of 3-haloacrylate. The observation that cis-CaaD requires two additional residues raises the question of how CaaD carries out a comparable reaction with fewer catalytic residues. As part of an effort to determine the basis for the apparently simpler CaaD-catalyzed reaction, the kinetic mechanism was determined by stopped-flow and chemical quench techniques using a fluorescent mutant form of the enzyme, αY60W-CaaD, and trans-3-bromoacrylate as the substrate. The data from these experiments as well as bromide inhibition studies are best accommodated by a six-step model that provides individual rate constants for substrate binding, chemistry, and a proposed conformational change occurring after chemistry followed by release of malonate semialdehyde and bromide. The conformational change and product release rates are comparable and together they limit the rate of turnover. The kinetic analysis and modeling studies validate the αY60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the αY60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated αY60W-CaaD mutant set the stage for an analysis of active site mutants to explore the contributions of individual catalytic residues and the basis for the simplicity of the reaction.

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3–C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD­(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2′-deoxy and 2′-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2′-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is L-arabinuronic acid with an apparent k cat of 3.0 s −1 . The product, L-glycero-L-galacto-octuluronate-1-phosphate, has a k cat /K m value of 2.1 × 10 3 M −1 s −1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of L-glycero-L-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for L-glycero-L-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for L-glycero-L-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-D-glycero-α-D-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is D-glycero-L-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-D-glycero-α-Dmanno-heptose to GDP-D-glycero-4-keto-α-D-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-D-glycero-4-keto-β-L-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-D-glycero-β-L-gluco-heptose. The threedimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-D-glycero-β-L-glucoheptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/ reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.