title: Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.
abstract: Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.
authors.affiliation: Conway Institute, UCD School of Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin 4, Republic of Ireland.
authors.forename: Nayla
authors.initials: N
authors.lastname: Munawar
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019617:Halobacterium salinarum / Q000201:enzymology* / Q000235:genetics*; D019622:Haloferax volcanii / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase