title: Improving thermal stability of thermophilic L-threonine aldolase from Thermotoga maritima.
abstract: Threonine aldolase (TA) catalyzes a reversible reaction, in which threonine is decomposed into glycine and acetaldehyde. The same enzyme can be used to catalyze aldol reaction between glycine and a variety of aromatic and aliphatic aldehydes, thus creating various alpha-amino-alcohols. Therefore, TA is a very promising enzyme that could be used to prepare biologically active compounds or building blocks for pharmaceutical industry. Rational design was applied to thermophilic TA from Thermotoga maritima to improve thermal stability by the incorporation of salt and disulfide bridges between subunits in the functional tetramer. An activity assay together with CD analysis and Western-blot detection was used to evaluate mutants. Except one, each of the designed mutants preserved activity toward the natural substrate. One of the 10 proposed single point mutants, P56C, displayed significantly enhanced stability compared to the wild type (WT). Its initial activity was not affected and persisted longer than WT, proportionally to increased stability. Additionally one of the mutants, W86E, displayed enhanced activity, with stability similar to WT. Higher activity may be explained by a subtle change in active site availability. Salt bridge formation between glutamic acid at position 86 and arginine at position 120 in the neighboring chain may be responsible for the slight shift of the chain fragment, thus creating wider access to the active site both for the substrate and PLP.
authors.affiliation: Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland; Laboratory of Theory of Biopolymers, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland. Electronic address: l.wieteska@yahoo.com.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Wieteska
authors.affiliation: Department of General Biophysics, University of Lodz, ul. Pomorska 141/143, 90-236 Lodz, Poland. Electronic address: maksion@biol.uni.lodz.pl.
authors.forename: Maksim
authors.initials: M
authors.lastname: Ionov
authors.affiliation: Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland. Electronic address: jszemraj@csk.umed.lodz.pl.
authors.forename: Janusz
authors.initials: J
authors.lastname: Szemraj
authors.affiliation: c-LEcta GmbH, Perlickstraße 5, 04-103 Leipzig, Germany. Electronic address: claudia.feller@c-lecta.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Feller
authors.affiliation: Laboratory of Theory of Biopolymers, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland. Electronic address: kolinski@chem.uw.edu.pl.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Kolinski
authors.affiliation: Laboratory of Theory of Biopolymers, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland. Electronic address: dgront@gmail.com.
authors.forename: Dominik
authors.initials: D
authors.lastname: Gront
mesh_terms: D020134:Catalytic Domain; D004795:Enzyme Stability / Q000502:physiology; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D012696:Glycine Hydroxymethyltransferase
keywords: Bioshell; Circular dichroism analysis; Mutagenesis; Stability improvement; Threonine aldolase