Characterization of Temperature Dependent Activity in a Model Polyextremophilic Beta-galactosidase Enzyme Through Kinetic and Structural Analysis

Abstract

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for polyextremophilic enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. Double-mutated enzymes were constructed and tested for temperature effects from 0-50 ºC, including two new tandem residue pairs, two potential loop insertions, and pairwise combination of the single residue mutations. The observed temperature and salinity dependent kinetic effects were compared to root-mean-square fluctuations to determine structural mechanisms of polyextremophilic activity. All the mutated enzymes were found to be more catalytically active at higher and/or less active at colder temperatures compared to the wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface-exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface-exposed residue, I476V, was paired with N251D or V482L, primarily a kcat effect was observed. The temperature dependent kinetic effects were continued to 25-50 ºC with a dominant Km effect observed in all mutated enzymes. By deleting the identified insertions/loops, we are able to determine the kinetic effects of these insertions. The domain B deletion resulted in a less active enzyme overall converging with the wild-type at higher temperatures while the domain C deletion resulted in reduced cold-activity and improved activity at higher temperatures. Domain B may confer cold-activity without changing the thermal stability while domain C confers cold activity, at least in part, at the expense of activity at higher temperatures. With molecular dynamics simulations, increased flexibility was observed with higher temperature and salinity and in the mutations, indicating that the enzyme’s function is related to its flexibility and there is a balance required for optimal polyextremophilic activity.