Analytical Biochemistry 544 (2018) 80–86

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Analytical Biochemistry

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A continuous assay for L-talarate/galactarate using circular T dichroism

∗ Nicole M. Eastona,1, Sarah A.E. Aboushawareba,1, Stephen L. Bearnea,b, a Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada b Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada

ARTICLE INFO ABSTRACT

Keywords: L-Talarate/galactarate dehydratase (TGD) is a member of the superfamily of and catalyzes the Dehydratase dehydration of either meso-galactarate or L-talarate to form 5-keto-4-deoxy-D-glucarate (5-KDG). To facilitate Assay study of this and other galactarate , a continuous circular dichroism-based assay has been Circular dichroism developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed Kinetics conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The m-Galactarate 2 −1 apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ± 2 deg cm dmol , which 5-Keto-4-deoxy-D-glucarate was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters −1 4 −1 −1 Km, kcat, and kcat/Km were 0.38 ± 0.05 mM, 4.8 ± 0.1 s , and 1.3 ( ± 0.2) × 10 M s , respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry 46, 9564–9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant

(Ki = 10.7 ± 0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.

Introduction aldonic and aldaric acid substrates. These features of the subgroup, and indeed the superfamily, have made annotation of function difficult 1 L-Talarate/galactarate dehydratase (EC 4.2.1.42, TGD ) is a member [8–10] and numerous detailed studies to understand the structure- of the mandelate racemase (MR) subgroup of the . function relationships among the members of the MR subgroup have Members of the MR subgroup share with other members of the enolase focused on delineating the specificity of the enzymes superfamily a common bidomain structure (i.e., (β/α)7β-barrel domain [5–7,11–20]. In addition to delineating the biochemical roles of dehy- and an α+β capping domain) and a common partial reaction (i.e., the dratases in metabolism, these enzymes have received at- metal-assisted, Brønsted base-catalyzed abstraction of the α-proton tention in recent years because the dehydratase-catalyzed formation of from a carboxylate substrate to form an enol(ate) intermediate) [1–4]. 2-ketoaldaric acids affords a route to deoxygenating in TGD catalyzes the dehydration of either meso-galactarate or L-talarate the biomass for use as biofuels, and to producing precursors for the to form 5-keto-4-deoxy-D-glucarate (5-KDG) as illustrated in Scheme 1 synthesis of value-added chemicals [21,22]. Such studies require con- [5]. Despite the fact that the MR subgroup is named for its archetype venient assays for the enzymes, preferably ones that are direct and MR, most enzymes in the subgroup, such as TGD, catalyze a dehydra- continuous. Several assays have been developed for galactarate dehy- tion reaction of a sugar acid substrate rather than a racemization re- dratases, including a coupled assay that utilizes 5-keto-4-deoxy-D-glu- action [6,7]. Although the catalytic machinery responsible for over- carate aldolase and L-lactate dehydrogenase as the coupling enzymes coming the kinetic and thermodynamic barriers accompanying [5], and fixed-time assays that involve detection of the through deprotonation of a weak carbon acid substrate and the overall struc- derivatization with semicarbazide [12,16,23] or treatment with peri- tural fold have been conserved within the MR subgroup, divergent odate and thiobarbituric acid [24]. In addition, the enzyme-catalyzed evolution has led to a variety of dehydratases that act on specific hydrogen-deuterium exchange at the α-position of the substrate has

Abbreviations: CD, circular dichroism; (His)6, N-terminal hexahistidine tag; 5-KDG, 5-keto-4-deoxy-D-glucarate; MR, mandelate racemase; TGD, L-talarate/galactarate dehydratase; StTGD, TGD from Salmonella typhimurium; Tris, tris(hydroxymethyl)aminomethane ∗ Corresponding author. Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada. E-mail address: [email protected] (S.L. Bearne). 1 Both authors contributed equally to the work. https://doi.org/10.1016/j.ab.2017.12.015 Received 29 August 2017; Received in revised form 30 November 2017; Accepted 11 December 2017 Available online 14 December 2017 0003-2697/ © 2017 Elsevier Inc. All rights reserved. N.M. Easton et al. Analytical Biochemistry 544 (2018) 80–86

Scheme 1. StTGD-catalyzed dehydration of m-galactarate and L-talarate to yield 5-KDG. been used to follow the reaction by 1H NMR spectroscopy [5,12] and respectively. The amplification parameters were adapted from those polarimetry has been used to follow the change in optical rotation reported by Gerlt and co-workers [5] as follows: initial denaturation at [5,12,16]. Some of these assay methods have their own particular 98 °C for 30 s, 40 cycles of 94 °C for 60 s for denaturation, thermal disadvantages. For example, the coupled assay is limited by the fact that gradient of 45–60 °C for 75 s for annealing, 68 °C for 120 s for extension, the aldolase is not readily available, specific conditions must be met for and 72 °C for 144 s for final extension. The amplification product, optimal functioning of the coupling enzymes, and the assay may not be purified using a QIAquick Gel Extraction Kit (Qiagen, Toronto, ON), amenable to inhibition studies because of the potential for structural and the pET-15b vector were then digested separately in a stepwise similarity of inhibitors to the substrate for the coupling enzyme. Neither manner with NdeI for 2 h followed by BamHI for 2 h at 37 °C, according the fixed-time assays nor the 1H NMR-based assay permit rapid de- to the manufacturer's instructions. Subsequently, the digestion products termination of initial rates, and the latter may have a solvent kinetic were purified as mentioned above and ligation with T4 DNA isotope effect when the reaction is conducted in D2O. However, re- (Invitrogen/Thermo Fisher, Waltham, MA) was conducted overnight at cognition that m-galactarate has the meso configuration and is optically 16 °C following the manufacturer's instructions. 25 inactive, while the product 5-KDG is optically active (αD = +45°) The pET-15b-StTGD plasmid encodes a fusion protein bearing an N- [5], led us to develop a circular dichroism (CD)-based assay for TGD terminal His6-tag. Chemically-competent E. coli DH5α cells were activity. This method is related to the polarimetric assay and could be transformed with the plasmid using heat shock, and glycerol stocks beneficial if a polarimeter is not available. Using the TGD from Sal- were prepared and stored at −80 °C using standard protocols [25]. The monella typhimurium (StTGD) and m-galactarate as the substrate, we sequence of the insert was verified by commercial DNA sequencing show that the CD-based assay offers a quick, practical, and effective (Robarts Research Institute, London, ON) to ensure incorporation of the method for monitoring TGD activity. insert and the absence of any mutations. Competent E. coli BL21 (DE3) cells were also transformed with the pET-15b-StTGD plasmid to be used Materials and methods for protein expression, and glycerol stocks were prepared and stored at −80 °C. General Enzyme purification Tartronic acid was obtained from Alfa Aesar (Tweksbury, MA). Mucic acid (m-galactaric acid) and all other chemicals were purchased StTGD was overexpressed and purified using a protocol similar to from Sigma-Aldrich Canada Ltd. (Oakville, ON). Synthetic deoxy-oli- that described in the Novagen manual [26]. Four starter cultures, each gonucleotide primers were purchased from Integrated DNA containing sterile Luria-Bertani (LB) medium (5 mL), ampicillin (25 μg/ Technologies (Coralville, IA). Restriction endonucleases were pur- mL), and the glycerol stock (10 μL), were incubated overnight at 37 °C chased from New England Biolabs (Ipswich, MA). An S1000 Thermal with continuous shaking at 250 rpm. These starter cultures were then Cycler (Bio-Rad Laboratories, Mississauga, ON) was used for poly- added to sterile LB medium (10 mL per 1 L) containing ampicillin merase chain reactions (PCR). A Branson Sonifier 250 was used for (25 μg/mL), which was then incubated for 2 h at 37 °C followed by sonication. His·Bind resin was purchased from Novagen (Madison, WI). 24 h at 27 °C (without induction by isopropyl β-D-1-thiogalactopyrano- All other chemicals were reagent grade or better. Assays were con- side (IPTG) [5]) with continuous shaking at 250 rpm. The cells were ducted using a Jasco J-810 spectropolarimeter (Jasco, Inc., Easton, harvested using centrifugation (3000 × g, 10 min, 4 °C). The cell pellet MD). 1H NMR analyses were conducted using a Bruker AV-500 spec- was re-suspended in 30 mL of ice-cold binding buffer [Tris-HCl buffer trometer at the Nuclear Magnetic Resonance Research Resource (NMR3, (20 mM, pH 7.9) containing NaCl (0.5 M) and imidazole (5 mM)]. Fol- Dalhousie University). lowing sonication on ice (5 × 30 s bursts with cooling for 1 min be- tween bursts), the cell lysate was then clarified by ultracentrifugation Cloning (110,000 × g, 30 min, 4 °C), and then passed through a Ni2+-charged His·bind resin-packed column (10-mL columns packed to 2.5 mL) at The open reading frame encoding StTGD (GI: 16766982) was cloned 4 °C. The column was subsequently washed with binding buffer from genomic DNA of Salmonella typhimurium LT2 using Phusion High- (25 mL), wash buffer [15 mL, Tris-HCl (20 mM, pH 7.9) containing NaCl Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). The (0.5 M) and imidazole (60 mM)], and strip buffer [7 mL, Tris-HCl PCR-based amplification reaction was conducted as reported by Gerlt (20 mM, pH 7.9) containing NaCl (0.5 M) and EDTA (100 mM)]. The and co-workers [5] using the forward (5′-GTGATTATCAGGAGAAAAC majority of the protein eluted in the strip buffer and the purity was ATATGGCTTTAAGCGCGAATTCCG-3′) and reverse (5′-GATTCCCGCCA assessed using 10% acrylamide SDS-PAGE followed by staining with GGATCCTTAAGGGCGTTTGCCAAATTCAC-3′) primers indicated, where Coomassie blue R-250. StTGD was then dialyzed against assay buffer the underlined bases correspond to NdeI and BamHI recognition sites, [Tris-HCl (50 mM, pH 8.0) containing MgCl2 (10 mM)] for 24 h at 4 °C

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