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Kinetic Characterization of 3‐Hydroxyisobutyrate Dehydrogenase from Arabidopsis thaliana
Author(s) -
Binkley Christiana,
McCool Mason,
Maurer Taylor,
Rouhier Kerry A
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.626.7
Subject(s) - biochemistry , valine , arabidopsis thaliana , amino acid , enzyme , nad+ kinase , biology , dehydrogenase , escherichia coli , hypothetical protein , catabolism , nucleotide , chemistry , mutant , gene
Branched‐chain amino acids (BCAAs) initially share a similar metabolic pathway. However, their paths diverge, resulting in unique catabolic pathways for each BCAA. This lab examined valine degradation, as it is the least characterized of all the BCAAs. During valine metabolism, the enzyme 3‐hydroxyisobutyrate dehydrogenase (HIBDH) catalyzes 3‐hydroxyisobutyric acid into methylmalonic semialdehyde as one of the concluding reactions. While HIBDH from Pseudomonas denitrificans had previously been cloned and expressed in Escherichia coli , the goal of this research was to examine the activity of HIBDH in plants. Thus, we cloned the HIBDH gene from Arabidopsis thaliana into E.coli , where its activity could be examined. Two HIBDH constructs were created in order to determine proper exclusion of the mitochondrial leader sequence; these constructs differ only by the length of their amino acid sequences. The expressed proteins were then purified by affinity chromatography and kinetically characterized. The construct composed of nucleotides 97–1044 (denoted At316) resulted in ~9 mg/mL of purified protein, while the construct generated from nucleotides 115–1044 (denoted At310) produced ~1 mg/mL from a similar volume and density of cells at the point of induction. Both constructs resulted in active protein using S‐HIBA (native substrate) and NAD, although very little activity was seen using construct At310. Construct At316 functioned optimally under alkaline conditions similar to HIBDH from P. denitrificans and Homo sapiens . Overall, the biochemical properties of HIBDH in A. thaliana were assessed, offering new insights into both the valine degradation pathway as a whole, as well as the biological and chemical parameters necessary for HIBDH function in plants. Support or Funding Information This project was funded by Kenyon College.