Part:BBa_K2710005
His-IaaH-SpyTag
6xHis IaaH with Spy-Tag
Usage and Biology
Indole-3-acetamide hydrolase is an enzyme involved in the biosynthesis of indole-3-aectic acid.
The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development1. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway2. The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway3. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid4.
Indole-3-acetamide hydrolase originating from Alcaligenes sp. strain HPC127114 was fused with a SpyTag and a hexahistidine tag (his tag).
The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins5. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from Streptococcus pyogenes to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide15. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher15, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements8.
It is particularly useful because neither component needs to be at the C or N terminus8, and the effect on the attached protein’s activity appears to be negligible10. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”9.
A 6x his tag (six consecutive histidine residues) was added to IaaH to enable purification using Immobilised Metal Affinity Chromatography (IMAC) columns6.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1128
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 661
- 1000COMPATIBLE WITH RFC[1000]
References
- Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! , (Springer Netherlands, 2007).
- Spaepen, S., Vanderleyden, J. & Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 31, 425-448, doi:10.1111/j.1574-6976.2007.00072.x (2007).
- Gaweska, H. M., Taylor, A. B., Hart, P. J. & Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. Biochemistry 52, 2620–6 (2013).
- Mishra, P., Kaur, S., Sharma, A. N. & Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. PLoS One 11, e0159009 (2016).
- Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).
- Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. & Stüber, D. Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Nat. Biotechnol. 6, 1321–1325 (1988).
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