Future Directions
Sequence Alignment.
Bower et al have indicated that threonine dehydrogenases can interact with a second enzyme, 2-amino-3-ketobutyrate CaA ligase (KBL), in order to catalyze the conversion of threonine to glycine [10]. A stable complex may form upon docking of the two proteins, shielding the highly unstable intermediate product 2-amino-3-ketobutyrate. This product is known to spontaneously and rapidly breakdown into aminoacetone and carbon dioxide [10]. As TM0436 has at present failed to show any enzyme activity, perhaps a coupled enzyme assay is required. To perform this, the correct binding partner of TMO436 must be found and procured. Sequence alignments will be performed against KBL to establish its commercial availability, and to find if a homolog has been characterized in T. maritima. Once obtained, the enzyme will be added the activity assay mixtures along with TM0436, to determine if activity can be observed.
Threonine->KBL->Glycine.
A problem with this solution may be suggested by Tressel et al, who mention that without KBL present, TDH causes the oxidative decarboxylation of threonine directly to aminoacetone [11]. Even without the KBL, the enzyme should display some activity with an A280 absorbance assay, as NAD+ would still be reduced. No noticeable activity suggests that threonine may not actually be the correct substrate for TM0436, despite sequence and homology data that suggest otherwise.
For this reason, the protein will be assayed against a variety of alcohol substrates, each under different conditions. First, a new batch of protein will be expressed and purified. Some samples will be dialyzed with EDTA to chelate metal ions native to the E. Coli vector, while some sample will be dialyzed without EDTA. Next the purified protein will undergo absorbance assays under varying temperature conditions, from 40-90° C, following the addition of zinc chloride and an alcohol to the assay cuvette. If activity is still elusive, the assays will be repeated using NADP+ as cofactor instead of NAD+.
Absorbance Assay using Spectrophotometer.
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