Lactobacillus reuteri 2'-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides

Abstract

A novel type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40 degrees C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2'-fluorodeoxyribonucleosides was carried out.

FIG. 1.

2′-Deoxyribosyltransferase reaction catalyzed by NDTs. E, enzyme; B₁ and B₂, purine or pyrimidine.

FIG. 2.

Purification of LrNDT. SDS-PAGE of different purification steps. Lane 1, 20 μg of total protein after Econo-Pac High Q cartridge chromatography. Lane 2, 10 μg of protein after size-exclusion chromatography on Superose 12. Lane P, prestained standard proteins from Bio-Rad used as molecular mass markers. The N-terminal amino acid sequence of the LrNDT subunit is shown in single-letter code.

FIG. 3.

Analytical ultracentrifugation analysis for LrNDT. Sedimentation experiments were carried out using a protein concentration of 0.675 mg/ml in 50 mM potassium phosphate buffer (pH 7)-0.5 M NaCl, at 20°C and 50,000 × g. (A) Sedimentation coefficient distribution c(s). (B) Sedimentation equilibrium gradient. The solid lines show the corresponding best-fit gradient for a single sedimenting species at sedimentation equilibrium with a bM_w value of 28,901 ± 149 Da.

FIG. 4.

Spectroscopic characterization of purified recombinant NDT from Lactobacillus reuteri. Far-UV CD spectra of LrNDT (0.21 mg/ml) in 50 mM potassium buffer (pH 7.0) at 25°C. (Inset) Thermal unfolding of LrNDT monitored by CD variation at 220 nm between 25 and 85°C scanned at 20°C/hour.

FIG. 5.

Temperature and pH dependence of LrNDT activity. (A) Thermal deactivation profile at 20°C, 30°C, 40°C, and 50°C (▪); 60°C (•); 65°C (▴); 70°C (□); 75°C (○); and 80°C (▵). Residual activity, a, of LrNDT was measured at 40°C under standard conditions. (B) Effect of temperature on the LrNDT activity (•) and stability (▵). (C) LrNDT stability at different pHs. (D) Optimal pH for LrNTD activity. The ionic strength (I) at each pH was adjusted to 150 mM by addition of NaCl.

FIG. 6.

Hypothetical active site structure of LrNDT. The active center of LrNDT has been determined by alignment of different 2′-deoxyribosyltransferase amino acid sequences of Lactobacillus strains. The program used was CLUSTAL W of the Biology Work Bench Server (31). A, ara-uracil (araU); B, 2′-fluoro 2′-deoxyuridine.