The cold-adapted β-galactosidase (R-β-Gal) from psychrotrophic bacterium Rahnella sp. R3 is a member of the glycoside hydrolases family 42 and it is a cold-adapted β-galactosidase. It was cloned and expressed in E. coli BL21. The purified R-β-Gal retained 80% of its maximum activity at temperatures as low as 4°C and formed a 225kDa trimeric structure in solution. R-β-Gal was crystalized and its crystal structure was solved. To the best of our knowledge, this is the first cold-adapted protein structure of GH42 enzymes. Each R-β-Gal molecule consists of three domains, an N-terminal catalytic domain that forms a (β/α)8 TIM barrel, a mixed β-sheet and α-helix domain, and a C-terminal β-sandwich domain. R-β-Gal did not require the presence of metal ions to be active and the presence of metal ions, such as Ca2+, Co2+ and Al3+ caused different levels of secondary changes of the enzyme. In consistent with the other structural known β-galactosidases of GH42, there is a zinc atom ligated by four cysteine residues in the second domain of R-β-Gal. Two residues, Glu157 and Glu314, superimposed well with the catalytic residues of other β-galactosidases were predicted to be required for its catalytic activity and this was verified by site-directed mutagenesis.

Cold-adapted β-galactosidase from a psychrotrophic bacterium Rahnella sp. R3: Protein structure and enzymatic properties

251st National Meeting (2016)

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