Double mutations far from the active site affect cold activity in an Antarctic halophilic β‐galactosidase

Abstract

The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for cold-active 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 temperaturedependent catalytic activity profiles via effects on K m and/or k cat compared to the wild-type enzyme. In this follow-up study, double-mutated enzymes were constructed and tested for temperature effects, including two new tandem residue pairs (N180T/A181T and T383A/S384A), and pairwise combination of the single residue mutations (N251D, F387L, I476V, and V482L). All doublemutated enzymes were found to be more catalytically active at moderate and/or less active at colder temperatures than wild-type, with both K m and k cat effects observed for the two tandem mutations. For pairwise combinations, a K m 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 mutation I476V located in a coiled region of domain B 25 Å from the active site was paired with N251D or V482L, a k cat effect was observed. These results indicate that temperature-dependent kinetic effects may be complex and subtle and are mediated by a combination of a small number of residues distant from the active site via changes to the hydration shell and/or perturbation of internal packing.

Effects of Salinity and Temperature on the Flexibility and Function of a Polyextremophilic Enzyme

“…This remarkable characteristic is a hallmark of haloarchaeal proteins and represents a primary mechanism of adaptation to the internal salinity. Genome-wide analysis also established the likely adaptive characteristics to low temperatures, which include subtle changes to the amino acid sequence distal to the active site and key surface residues [ 16 ].…”

“…This suggests the salinity effect on the catalytic rate is influenced by the “softness” as it is described by Aqvist et al, or flexibility, of the surface of the protein [ 21 ]. Our previous mutagenesis studies have shown that residues distant from the active site can have an effect on the catalytic rate of this polyextremophilic protein [ 15 , 16 ]. This is similar to the effects seen with temperature with cold-adapted proteins in general having more surface flexibility [ 23 ].…”

“…Site-directed mutagenesis and steady-state kinetic studies were conducted which found five of the amino acid residues to be important for enhanced cold activity or reduced activity at moderately high temperatures. A series of double mutations showed that key residues with substantial surface exposure distal from the active site (16–48 Å) were dominant [ 16 ]. These results indicated that temperature-dependent kinetic effects were complex and subtle and are likely mediated by changes to the hydration shell and/or perturbation of internal packing.…”

β‐Galactosidase: Insights into source variability, genetic engineering, immobilisation and diverse applications in food, industry and medicine

“…Laye and DasSarma have discovered that making modifications far from the active site of H. lacusprofundi b-Galactosidase could lead to an increase in the activation temperature, while modifications near to the active site could allow the enzyme to function at low temperatures due to the increased flexibility of its molecules. The change in flexibility may protect the active site for that the enzyme has a space and thus metabolise the molecule (Laye and DasSarma 2022).…”