A combination of bioinformatic and experimental approaches are being used for understanding protein function and adaptation in extremophilic microorganisms of the Family Haloarchaea (1). Best reciprocal blast analysis was initially used to identify nearly 800 conserved proteins, cHOGs (conserved Haloarchaeal Orthologous Groups), encoded by all sequenced genomes in these salt and stress tolerant microorganisms (2). Functions were assigned to 89% of proteins, including metabolism, information transfer and storage, cell processes and signalling, and poorly characterized/general function groups. To address protein adaptation to cold temperature, we determined which amino acid residues were substituted in a cold-adapted H. lacusprofundi species from Antarctica at positions conserved in orthologous proteins from warm temperature haloarchaea (3). Our results showed that proteins from the Antarctic species have subtle alterations in amino acid size and/or polarity, consistent with increased protein flexibility and improved function at colder temperatures. For the cold-active �-galactosidase from H. lacusprofundi, we focused on amino acid substitutions in the context of a model structure at positions invariant in warm-active homologs. In surface regions of the enzyme, substitutions of charged amino acids with neutral residues or increased hydrophobicity were observed, while in interior regions of the enzyme, subtle changes in amino acid size and polarity were found. To test the importance of the observed amino acid substitutions in cold-activity, the H. lacusprofundi �-galactosidase gene was cloned and overexpressed in our experimental haloarchaeal model organism, Halobacterium sp. NRC-1, purified to homogeneity, and characterized biochemically (4). Our recent efforts are directed at mutating these key amino acid residues in the Antarctic �-galactosidase to establish their roles in function under extreme conditions.1. Karan, R. et al. 2012. Aquatic Biosystems8:4.2. Capes, M.D. et al. 2012. BMC Genomics13:39.3. DasSarma, S. et al. 2013. PLoS ONE8:e58587.4. Karan, R. et al. 2013. BMC Biotechnology 13:3.

Combined bioinformatic and experimental approach to extreme enzyme function:  The case of a cold and salt active �-galactosidase from Antarctica

248th National Meeting (2014)

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