title: Identification and characterization of protein encoded by orf382 as L-threonine dehydrogenase.
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 cel ls 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 micrometer), ethanol (222.5 micrometer), and n-butanol (8.02 micrometer) 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%).
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ma
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wang
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Ma
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D013912:Threonine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase