2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity

Abstract

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.

Keywords: 2-keto-3-deoxygluconate; 2-ketogluconate; 2-ketogluconate kinase; 2-ketogulonate; Cupriavidus necator; biocatalysis; monosaccharides phosphate.

Figure 1

Reaction catalyzed by 2-ketogluconate kinase (KGUK).

Figure 2

Substrate kinetics of kinase activity for KGUK from C. necator for different substrates (at 25 °C). Activity of purified KGUK was measured at increasing concentrations of the acceptor substrates 2-keto-D-gluconate (KG) (A), 2-keto-3-deoxygluconate (KDG) (B), 2-ketogulonate (KGul) (C), and 2-keto-3-deoxygulonate (KDGul) (D), maintaining a constant excess of ATP (1.25 mM) and Mg²⁺ (5 mM). Final concentration of pure KGUK was customized in each kinetic experiment to optimize the activity measurement: 2.5 μg/mL of purified enzyme for KG kinetics (A), 1.5 μg/mL for KDG assays (B), and 3.4 μg/mL for KGul and KDGul experiments (C and D).

Figure 3

Kinase activity of the purified KGUK from C. necator was measured at increasing concentrations of ATP, maintaining a constant excess of KG (1.25 mM) as the phosphate acceptor and 2.1 μg/mL of purified enzyme. ATP kinetics were carried out for kinetic constants calculation using a Mg²⁺ excess concentration of 5 mM (A). To evaluate the effect of higher concentrations of ATP on the enzyme activity, additional assays were performed increasing the Mg²⁺ concentration to 25 mM (B). When ATP concentrations over 5 mM were used, a strong decrease in the kinase activity was observed.

Figure 4

2-keto-carboxylic acid sugars tested as KGUK_Cnec substrate.

Figure 5

Sequence alignments and substrate accommodations of the concerned residues. (A) Amino acid sequence alignments of KGUK from C. necator (1.) and 2-keto-3-deoxy-d-gluconate kinase (KDGK) from Thermus thermophilus (2.). Grey-shaded amino acids have been identified as active site residues in the crystallographic structure of the KDGK enzyme. Only three residues (shaded in black) are different in the C. necator enzyme. (B) Structure of the active site of KDGK from T. thermophilus.

Figure 6

Reaction catalyzed by the KDGK enzymes.

Figure 7

Phosphorylation reactions: analytical (assay) and practical (synthesis) scale. (A) KGUK activity assay. Substrate phosphorylation was measured with a coupled enzymatic system, where the decrease of NADH absorbance at 340 nm was directly proportional to substrate phosphorylation. PK: pyruvate kinase, PEP: phosphoenolpyruvate, pyr: pyruvate, lact: lactate, LDH: lactate dehydrogenase. (B) Biocatalytic orthogonal cascade for the synthesis of 2-ketogluconate-6-phosphate (KGP).

Figure 7

Phosphorylation reactions: analytical (assay) and practical (synthesis) scale. (A) KGUK activity assay. Substrate phosphorylation was measured with a coupled enzymatic system, where the decrease of NADH absorbance at 340 nm was directly proportional to substrate phosphorylation. PK: pyruvate kinase, PEP: phosphoenolpyruvate, pyr: pyruvate, lact: lactate, LDH: lactate dehydrogenase. (B) Biocatalytic orthogonal cascade for the synthesis of 2-ketogluconate-6-phosphate (KGP).

Figure 8

Optimization of the KG/PEP ratio in different KG phosphorylation reactions with ATP regeneration. (A) Reactions were performed in 1 mL of Tris-HCl buffer 50 mM and 50 μmol of the limiting substrate at different ratios of KG/PEP (1.0/0.5 (●), 1.0/0.7 (○), and 1.0/0.9 (▼), 1.0/1.1 (∆)) and a control reaction with no ATP (■). (B) Reactions were performed in 2 mL of Tris-HCl buffer 50 mM and 40 μmol of the limiting substrate at different ratios of KG/PEP (1.0/0.5 (●) and 1.0/0.7 (○)) and a control reaction with no ATP (■). (C) Reactions were performed in 2.5 mL of Tris-HCl buffer 50 mM and 35 μmol of the limiting substrate employing a KG/PEP ratio of 1.0/0.8.