Structural insights into the substrate stereospecificity of d-threo-3-hydroxyaspartate dehydratase from Delftia sp. HT23: a useful enzyme for the synthesis of optically pure l-threo- and d-erythro-3-hydroxyaspartate

Abstract

D-threo-3-Hydroxyaspartate dehydratase (D-THA DH) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme, isolated from a soil bacterium of Delftia sp. HT23. It catalyzes the dehydration of D-threo-3-hydroxyaspartate (D-THA) and L-erythro-3-hydroxyaspartate (L-EHA). To elucidate the mechanism of substrate stereospecificity, crystal structures of D-THA DH were determined in complex with various ligands, such as an inhibitor (D-erythro-3-hydroxyaspartate (D-EHA)), a substrate (L-EHA), and the reaction intermediate (2-amino maleic acid). The C (β) -OH of L-EHA occupied a position close to the active-site Mg(2+), clearly indicating a possibility of metal-assisted C (β) -OH elimination from the substrate. In contrast, the C (β) -OH of an inhibitor was bound far from the active-site Mg(2+). This suggests that the substrate specificity of D-THA DH is determined by the orientation of the C (β) -OH at the active site, whose spatial arrangement is compatible with the 3R configuration of 3-hydroxyaspartate. We also report an optically pure synthesis of L-threo-3-hydroxyaspartate (L-THA) and D-EHA, promising intermediates for the synthesis of β-benzyloxyaspartate, by using a purified D-THA DH as a biocatalyst for the resolution of racemic DL-threo-3-hydroxyaspartate (DL-THA) and DL-erythro-3-hydroxyaspartate (DL-EHA). Considering 50 % of the theoretical maximum, efficient yields of L-THA (38.9 %) and D-EHA (48.9 %) as isolated crystals were achieved with >99 % enantiomeric excess (e.e.). The results of nuclear magnetic resonance signals verified the chemical purity of the products. We were directly able to isolate analytically pure compounds by the recrystallization of acidified reaction mixtures (pH 2.0) and thus avoiding the use of environmentally harmful organic solvents for the chromatographic purification.

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

“…DL-EHA was synthesized by ammonolysis of (±)-trans-epoxysuccinic acid (Tokyo Chemical Industry, Tokyo, Japan) at 60°C as described previously. 12) The synthesis was confirmed by silica gel thin layer chromatography (TLC) using 7:1:2 (v/v/v) of ethanol/aqueous ammonia (28%)/distilled water as a developing solvent. L-THA, L-EHA, and DL-THA were purchased from Tocris Cookson, Ltd. (Bristol, UK), Wako Pure Chemical Industries (Osaka, Japan), and Tokyo Chemical Industry, respectively.…”

“…HT23. 12) Restriction endonucleases were obtained from TaKaRa Bio Inc. (Kusatsu, Japan). All other chemicals were of analytical grade and commercially available.…”

“…The activity of 3-hydroxyaspartate dehydratase was determined as described previously. 12) The enzyme activity was coupled to malate dehydrogenase, which reduces oxaloacetate formed by the dehydratase to malate in the presence of NADH. One unit of the enzyme was defined as the amount capable of catalyzing the oxidation of 1 μmol of NADH per min.…”

“…In contrast, a racemic mixture of DL-THA and DL-EHA can be easily synthesized from inexpensive cis-or trans-epoxysuccinate in a one-step reaction. 11,12) Thus, enzymes that can distinguish the L-form or D-form of 3-hydroxyaspartate isomers are useful for the enzymatic resolution of DL-racemic 3-hydroxyaspartate mixtures.…”

“…13) The production of optically active L-THA from DL-THA by enzymatic resolution using this enzyme has also been reported. 12) In addition, the enzymatic syntheses of L-THA from L-aspartate or L-asparagine have also been reported. 14,15) However, no dehydratase acting on the D-erythro form of 3-hydroxyaspartate has been reported so far.…”