1. L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity
Hui-Min Qin, et al. Acta Crystallogr D Biol Crystallogr. 2014 Jun;70(Pt 6):1695-703. doi: 10.1107/S1399004714007664. Epub 2014 May 30.
L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.
2. Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography
Hua Zhao, Kenji Hamase, Akiko Morikawa, Zongyin Qiu, Kiyoshi Zaitsu J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Oct 25;810(2):245-50. doi: 10.1016/j.jchromb.2004.08.006.
A sensitive and selective method for the determination of four threonine (Thr) isomers (L-Thr, D-Thr, L-allo-Thr and D-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of D-Thr, D-allo-Thr and L-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol-5 pmol per injection, and that of L-Thr is 50 fmol-50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of D-Thr, D-allo-Thr and L-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of D-Thr and D-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of D-Thr (0.85 +/- 0.05 nmol/g wet tissue) and D-allo-Thr (5.01 +/- 0.32 nmol/g wet tissue) were found in the corpus striatum. L-allo-Thr was not present in any of the tested tissues and physiological fluids.
3. Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39
M Kataoka, M Wada, K Nishi, H Yamada, S Shimizu FEMS Microbiol Lett. 1997 Jun 15;151(2):245-8. doi: 10.1111/j.1574-6968.1997.tb12577.x.
L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).
