Part:BBa_K4395014
SpyTag-AAS-YahK-SpyCatcher is a fusion protein whose thermostability is increased by the SpyTag-SpyCatcher tagging system while the catalytic activity not affected.
AAS(Aromatic acetaldehyde synthase),catalyzes the production of 4-hydroxyphenylacetaldehyde (HPAA) directly from L-tyrosine, tyramine not being formed as an intermediate[1].AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM)[2].In addition, this enzyme has been actively expressed in E.coli[3].These characteristics of AAS attract us and thus we choose it as Aromatic acetaldehyde synthase of our experiment.
NADPH-dependent aldehyde reductase YahK catalyzes the reduction of a wide range of aldehydes into their corresponding alcohols. Has a strong preference for NADPH over NADH as the electron donor. Cannot use a ketone as substrate. Is a major source of NADPH-dependent aldehyde reductase activity in E.coli[4]. In addition, this enzyme has been actively expressed in E.coli[5].These characteristics of RsAs attract us and thus we choose it as aldehyde reductase of our experiment.
The SpyTag/SpyCatcher system is a split-in-two of the immunoglobulin-like collagen adhesion domain of Streptococcus pyogenes denoted CnaB2. CnaB2 is characterized by an internal isopeptide bond between residue Lys31 and residue Asp117. When split in two, one of which containing Lys31 and another containing Asp117, they associate and spontaneously form the isopeptide bond, thus join together [6]
The composite part SpyTag-AAs-Yahk-SpyCatcher is a fusion protein cyclized by the SpyTag/SpyCatcher system, whose two components are fused on both ends. This cyclization process increases the thermostability of the enzyme, enabling catalysis in an unfavored heated system [7].
References:
1.Torrens-Spence MP, Gillaspy G, Zhao B, Harich K, White RH, Li J. Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme. Biochem Biophys Res Commun. 2012 Feb 10;418(2):211-6. doi: 10.1016/j.bbrc.2011.12.124. Epub 2012 Jan 11. PMID: 22266321.
2.Torrens-Spence MP, Liu P, Ding H, Harich K, Gillaspy G, Li J. Biochemical evaluation of the decarboxylation and decarboxylation-deamination activities of plant aromatic amino acid decarboxylases. J Biol Chem. 2013 Jan 25;288(4):2376-87. doi: 10.1074/jbc.M112.401752. Epub 2012 Nov 30. PMID: 23204519; PMCID: PMC3554908. 3.Trantas E, Navakoudis E, Pavlidis T, Nikou T, Halabalaki M, Skaltsounis L, Ververidis F. Dual pathway for metabolic engineering of Escherichia coli to produce the highly valuable hydroxytyrosol. PLoS One. 2019 Nov 4;14(11):e0212243. doi: 10.1371/journal.pone.0212243. Erratum in: PLoS One. 2019 Dec 13;14(12):e0226760. PMID: 31682615; PMCID: PMC6828502. 4.Pick A, Rühmann B, Schmid J, Sieber V. Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production. Appl Microbiol Biotechnol. 2013;97(13):5815-5824. doi:10.1007/s00253-012-4474-5 5.Kramer L, Le X, Rodriguez M, Wilson MA, Guo J, Niu W. Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy. ACS Synth Biol. 2020;9(7):1632-1637. doi:10.1021/acssynbio.0c00290 6.Long Li, Jacob O. Fierer, et al. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag J Mol Biol. 2014 January 23; 426(2): 309–317 7.Xiao-Bao Suna, Jia-Wen Caoa,b, et al. SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation
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