title: Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling.
abstract: A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T(opt)=68 degrees C), and the hyperthermophilic xylanase B (TmxB, T(opt)=102 degrees C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T(opt)=80 degrees C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N- and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N- and C-termini are in contact.
authors.affiliation: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Pf. 7, H-1518 Budapest, Hungary.
authors.forename: Szilárd
authors.initials: S
authors.lastname: Kamondi
authors.forename: András
authors.initials: A
authors.lastname: Szilágyi
authors.forename: László
authors.initials: L
authors.lastname: Barna
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases