title: Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method.
abstract: α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.
authors.affiliation: School of Chemical and Biomolecular Engineering, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-0363, USA.
authors.forename: Janna K
authors.initials: JK
authors.lastname: Blum
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Ricketts
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
mesh_terms: D000465:Algorithms; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D016384:Consensus Sequence; D004795:Enzyme Stability; D008954:Models, Biological; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature; D014974:Xanthomonas / Q000502:physiology*
chemical_list: D002265:Carboxylic Ester Hydrolases; C058058:alpha-amino acid esterase