Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase
| Autor: | Shiladitya DasSarma, Victoria J. Laye, Ram Karan, Priya DasSarma, Wolf T. Pecher, Jong-Myoung Kim |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Halobacterium
Models Molecular Protein Conformation alpha-Helical 0301 basic medicine Archaeal Proteins 030106 microbiology Antarctic Regions Gene Expression Crystallography X-Ray Substrate Specificity Structure-Activity Relationship 03 medical and health sciences Catalytic Domain TIM barrel Protein Interaction Domains and Motifs Homology modeling Enzyme kinetics Cloning Molecular Halorubrum Site-directed mutagenesis Psychrophile Multidisciplinary biology Active site Biological Sciences Nitrophenylgalactosides beta-Galactosidase biology.organism_classification Recombinant Proteins Enzyme assay Cold Temperature Kinetics 030104 developmental biology Amino Acid Substitution Biochemistry Mutagenesis Site-Directed biology.protein Haloarchaea Thermodynamics Protein Conformation beta-Strand Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 114:12530-12535 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1711542114 |
| Popis: | The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|