Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii

Phytochemistry. 2017 Mar;135:18-23. doi: 10.1016/j.phytochem.2016.12.012. Epub 2016 Dec 27.

Abstract

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.

Keywords: Chlamydomonadaceae; Chlamydomonas reinhardtii; Enzyme characterization; Green alga; Pyridoxal 5’-phosphate; d-Amino acid; d-Threonine aldolase.

MeSH terms

  • Aldehyde-Lyases / metabolism
  • Amino Acid Sequence
  • Chlamydomonas reinhardtii / enzymology
  • Chlamydomonas reinhardtii / metabolism*
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Glycine / metabolism
  • Glycine Hydroxymethyltransferase / metabolism*
  • Pyridoxal Phosphate / metabolism
  • Stereoisomerism
  • Substrate Specificity
  • Threonine

Substances

  • Threonine
  • Pyridoxal Phosphate
  • Glycine Hydroxymethyltransferase
  • Aldehyde-Lyases
  • threonine acetaldehyde-lyase
  • Glycine