Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate

Abstract

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.

FIGURE 1.

Reaction scheme of the first steps of the oxidative pathway for d-galacturonate catabolism. d-Galacturonate is oxidized by the uronate dehydrogenase, At Udh, to d-galactarolactone which can hydrolyze spontaneously or with the aid of a lactonase to meso-galactarate. Then a dehydratase converts galactarate to 3-deoxy-2-keto-hexarate. In the present work, we show that A. tumefaciens has an enzyme, At Gci, for the direct conversion of the d-galactarolactone to 3-deoxy-2-keto-l-threo-hexarate.

FIGURE 2.

SDS-PAGE of purified At Gci. Lane 1, molecular weight marker proteins in kDa; lane 2, native At Gci partially purified from A. tumefaciens using HIC, DEAE and Resource Q chromatography; and lane 3, At Gci expressed and purified from E. coli. The proteins were visualized using the Criterion Stain Free gel imaging system (Bio-Rad).

FIGURE 3.

Kinetic properties of the purified At Gci on d-galactaro- and d-glucarolactone. Kinetic data were obtained from incubations of 0.6 μg of enzyme with d-galactaro- or d-glucarolactone (0.05–15 mm) in sodium phosphate buffer pH 6.8, 5 mm MgCl₂at 22 °C. Inset: original data showing the measured initial rates in the substrate concentration area from 0- 10.0 mm. The apparent kinetic parameters were obtained by curve fitting analysis using Graphpad Prism software 4.01.

FIGURE 4.

A, 600 MHz ¹H NMR spectrum of a mixture of galactaric acid and d-galactaro-1,4-lactone in H₂O at 22 °C. The galactaric acid signals at about 3.9 and 4.2 ppm are marked by asterisks, while the lactone signals (two doublets at 4.5–4.6 ppm, a triplet 4.35 ppm, and a narrow doublet overlapping with the galactaric acid signal at 4.2 ppm) are unmarked. B, ¹H NMR spectrum of a complete reaction of the mixture of galactaric acid and d-galactaro-1,4-lactone with At Gci. The lactone signals, which were unmarked in the upper panel have disappeared, while the asterisk-marked galactaric acid signals remain unchanged. New signals arising from the product KDG have appeared at 4.3–4.6 ppm, 3.9 ppm, 3.0 ppm, and between 2.1 and 2.5 ppm. The intensity of the product signals is low, because several different forms of the product are present. C, ¹H NMR spectrum of a complete reaction of the mixture of galactaric acid and d-galactaro-1,4-lactone with the control enzyme TalrD/GalrD. In this reaction, the galactaric acid signals have disappeared, while the lactone signals remained unchanged. The signals of the product are identical with both of the enzymes. The large signal at 3.9 ppm in panels B and C originates from the Tris buffer of the enzyme preparations.

FIGURE 5.

A, 500 MHz ¹H NMR spectrum of KDG. The letters above the spectrum indicate the three different spin-systems identified from a COSY spectrum. The presence of three distinct spin systems indicates that KDG exists in three different forms. B, ¹³C NMR and DEPT135 spectra of 3-deoxy-2-keto-hexarate. In DEPT135 spectrum, CH₃ and CH carbons give positive signals, while CH₂ carbon signals are negative. Comparison of the ¹³C spectrum with the DEPT spectrum was used to identify the signals of the quaternary carbons which are not present in the DEPT spectrum.

FIGURE 6.

A, configuration of possible reaction products obtained by conversion of d-galactaro-1,4-lactone and d-glucaro-1,4-lactone by At Gci. B, configuration of the reaction products obtained in the reaction of At Gci with d-glucaro-1,4-lactone. The carbons involved in reaction are marked with a filled circle (●, C4 in the substrate) and a cross (x, C5 in the substrate). C3 in the product correspond to C4 in the substrate.

FIGURE 7.

A, expansion of a 500 MHz ¹H NMR spectrum of a mixture of d-glucaric acid and its 1,4- and 6,3-lactones in H₂O at 12 °C. The d-glucaro-1,4- and 6,3-lactone signals from which the substrate specificity of At Gci was observed are labeled as 1,4 and 6,3, respectively. B, ¹H NMR spectrum of the same sample after a complete reaction with At Gci. The signals of the d-glucaro-1,4-lactone have disappeared, while the signals of d-glucaro-6,3-lactone and d-glucaric acid remain unchanged. This indicates that only the 1,4-lactone is a substrate of the enzyme. The product gives signals identical to the ones obtained from the reaction with d-galactaro-1,4-lactone (Fig. 4B), although the exact chemicals shifts are different because of the lower measurement temperature.