D-allo-Threonine

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-β-d-Quip3N(d-allo-ThrAc)-(1→2)-β-d-Ribf-(1→4)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element β-d-Galp-(1→3)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.

The terahertz (THz) band contains a wealth of information about vibration and rotational energy levels, most of the vibration modes between amino acid molecules are in the THz band, so it reflects many unique absorption characteristics in the THz band. The use of terahertz time-domain spectroscopy can not only effectively identify different kinds of amino acids but also distinguish various isomers of the same amino acid due to the varied vibration modes. The absorption spectra of four stereoisomers of threonine were investigated by terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared spectroscopy. The results show that the isomers show similarity in the infrared band, while manifest evidently the similarity between enantiomers L-threonine and D-threonine, and between L-allo-threonine and D-allo-threonine, and the difference between diastereoisomer L-threonine/D-threonine and L-allo-threonine/D-allo-threonine in the terahertz band. In order to fully understand the origin of the terahertz absorption characteristics of isomer molecules, simulation calculations were carried out in combination with density functional theory to connect the vibrational modes and molecular structures, Furthermore, the unit cell configurations of L-thr and L-allo-thr and the differences between various low-frequency vibrational modes are analyzed from the perspective of hydrogen bond configuration. By further extracting terahertz optical parameters such as refractive index and imaginary part of the dielectric constant of threonine isomers, the results show that the refractive index spectrum and dielectric loss spectrum can clearly show the response characteristics of the orientation polarization of dipole inside threonine isomers in terahertz band.