Establishment of an efficient one-step enzymatic synthesis of cyclic-2,3-diphosphoglycerate

Abstract

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their considerable potential, only a limited number of extremolytes - most notably ectoine and hydroxyectoine - have achieved commercial relevance, primarily due to the absence of efficient production strategies for the majority of other extremolytes. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg²⁺, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a V_max of 38.2 U mg^-1, with K_M values of 1.52 mM for 2,3DPG and 0.55 mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100 mM, it achieved full conversion of 37.6 mg of 2,3DPG to cDPG within 180 min, using just 0.5 U of recombinant MfcDPGS at 55°C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

Keywords: 2-phosphoglycerate kinase; archaea; compatible solutes; cyclic-2,3-diphosphoglycerate synthetase; extremolytes; hyperthermophiles; stress response; thermoprotection.

Figure 1

Pathway for cDPG synthesis and structure of MfcDPGS dimer. (A) Schematic representation of the enzymatic synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by the formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate (2,3DPG). (B) Enzyme structure of the Methanothermus fervidus cDPGS dimer PDB 8ORU viewed perpendicular to its molecular dyad. The two subunits are shown in blue and light blue [visualized using CCP4mg (McNicholas et al., 2011)].

Figure 2

Purification of recombinant MfcDPGS expressed in Escherichia coli. The codon-optimized cdpgs gene was cloned into the pET15b vector without an affinity tag, and expression was optimized in Escherichia coli BL21(DE3)-Codon-Plus. The recombinant protein was then purified by heat precipitation and size exclusion chromatography (SEC). Protein samples (5–10 μg) from each purification steps were analyzed by SDS-PAGE (12.5%) and the gel was stained with Coomassie Brilliant Blue. CE: Crude extract, HP₇₅: Soluble fraction after heat precipitation (75°C, 30 min), SEC: Fraction after size exclusion chromatography. M: Protein marker, unstained protein ladder (Thermo Fisher Scientific, Carlsbad, USA).

Figure 3

Effect of salt concentration and pH on the activity of MfcDPGS. (A) Effect of KCl concentration (0, 100, 200, 400 mM) and (B) effect of pH (pH 5.5 to 8.0) on the activity of partially purified MfcDPGS (16.8 μg) after heat precipitation (75°C, 30 min). The enzyme activity was determined using the continuous PK-LDH assay at 55°C, which couples the formation of ADP from ATP to the oxidation of NADH monitored by the absorbance decrease at 340 nm. The pH optimum was determined using buffer systems as described in the Material and Method section, with each buffer supplemented with 400 mM KCl and 10 mM MgCl₂. The relative activity is expressed as a percentage, where 100% corresponds to a specific activity of 1.8 U mg⁻¹. All assays were performed in technical triplicates, and the error bars indicate the standard deviation of the mean.

Figure 4

Enzymatic properties of the recombinant MfcDPGS. Enzymatic activity (A,B) was measured at 55°C (340 nm) with 4.8 μg of purified enzyme using the continuous PK-LDH assay. (A) The specific activity of purified MfcDPGS was determined with varying concentration of 2,3DPG (0–10 mM) and a constant 10 mM ATP. (B) The specific activity was determined with varying concentrations of ATP (0–10 mM) and a constant 10 mM 2,3DPG. All assays were performed in technical triplicates, and the error bars represent the standard deviation of the mean. Computational fits to the complete dataset are shown as solid black lines and the mean prediction confidence intervals are represented as shaded areas.

Figure 5

Long term stability of the recombinant MfcDPGS. The enzyme was stored in aliquots at −70°C in a buffer containing MES/KOH (pH 6.5), 25% (v/v) glycerol, 400 mM KCl, 10 mM MgCl₂ and 10 mM DTT. Aliquots were thawed gently on ice and mixed thoroughly by pipetting before activity measurement. The residual activity of the thawed enzyme samples was determined at the indicated time points using the continuous PK-LDH assay. 100% of relative activity corresponds to a specific activity of 18.7 U mg⁻¹. All measurements were performed in technical triplicates, the error bars represent the standard deviation of the mean. Results are based on two biological replicates, each from a separate expression and purification run.

Figure 6

Conversion of 2,3DPG to cDPG by recombinant MfcDPGS. The time-dependent conversion results for (A) 1 mM (circles) and 10 mM (squares) conversion of 2,3DPG and (B) 25 mM (circles) and 100 mM (squares) 2,3 DPG to cDPG are shown. Assays were performed at 55°C in 50 mM MES/KOH (pH 6.5), 400 mM KCl and a Mg²⁺/ATP ratio of 0.2 using 0.5 U of purified MfcDPGS. Samples were taken at regular time intervals, and resulting ADP from ATP was quantified using the PK-LDH assay and are shown with blue open symbols. Each data point represents the mean value of three independent technical replicates. The redfilled symbols represent the 2,3DPG concentrations, while the blue filled symbols represent the cDPG concentrations, both quantified via ³¹P-NMR spectroscopy. Model (Equations 1, 2) predictions for the experiments are shown with a red line for 2,3DPG consumption and a blue line for cDPG production.