Identification and characterization of protein encoded by orf382 as L-threonine dehydrogenase

Fei Ma, Tianwen Wang, Xingyuan Ma, Ping Wang

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant 6×His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, L-threonine, ethanol, isopropanol, glucose, glycerol, L-serine, lactic acid, citric acid, methanol, or D-threonine), the enzyme showed the highest activity on L-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of L-threonine into glycine. Considering the presence of tested substrates in living E. coli cells and previous literature, we believed that the suitable nomenclature for the enzyme should be an L-threonine dehydrogenase (LTDH). When using L-threonine as the substrate, the enzyme exhibited the best catalytic performance at 39°C and pH 9.8 with NAD+ as the cofactor. The determination of the Km values towards L-threonine (Km = 11.29 μM), ethanol (222.5 μM), and n-butanol (8.02 μM) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the α-helix percentage (from 12.6% to 6.3%).

Original languageEnglish (US)
Pages (from-to)748-755
Number of pages8
JournalJournal of Microbiology and Biotechnology
Volume24
Issue number6
DOIs
StatePublished - 2014

Keywords

  • Alcohol dehydrogenase
  • Circular dichroism
  • Gene knockout
  • Identification
  • L-threonine dehydrogenase
  • RED recombination

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