Zinc Modulates Self-Assembly of Bacillus thermocatenulatus Lipase

Abstract

Thermoalkalophilic lipases are prone to aggregation from their dimer interface to which structural zinc is very closely located. Structural zinc sites have been shown to induce protein aggregation, but the interaction between zinc and aggregation tendency in thermoalkalophilic lipases remains elusive. Here we delineate the interplay between zinc and aggregation of the lipase from Bacillus thermocatenulatus (BTL2), which is taken to be a representative of thermoalkalophilic lipase. Results showed that zinc removal disrupted the BTL2 dimer, leading to monomer formation and reduced thermostability manifesting as a link between zinc and dimerization that leads to thermostability, while zinc addition induced aggregation. Biochemical and kinetic characterizations of zinc-induced aggregates showed that the aggregates obtained from the early and late stages of aggregation had differential characteristics. In the early stages, the aggregates were soluble and possessed native-like structures, while in the late stages, the aggregates became insoluble and showed fibrillar characteristics with binding affinities for Congo red and thioflavin T. The impact of temperature on zinc-induced aggregation was further investigated, and it was found that the native-like early aggregates could completely dissociate into functional lipase forms at high temperatures while dissociation of the late aggregates was limited. To this end, we report that the zinc-induced aggregation of BTL2 can be reversed by temperature switches and initiated by ordered aggregates in the early stages that gain fibrillar-like features over time. Insights revealed by this work contributes to the knowledge of aggregation mechanisms that exist in thermophilic proteins, reflecting the potential use of metal addition and/or removal to fine-tune aggregation tendency.

Understanding thermal and organic solvent stability of thermoalkalophilic lipases: insights from computational predictions and experiments

“…Although numerous studies have investigated thermal stability of lipase thermoalkalophilic lipases, the molecular machinery behind the organic solvent stability of this lipase family or whether there is a link between organic solvent and thermostability still remains elusive. [12][13][14] Particularly, discovery of any molecular links between thermal and organic solvent stability might ameliorate the rational design process, leading to the design of highly stable thermoalkalophilic lipase variants at elevated temperatures and in organic solvents.…”

Probing the roles of two tryptophans surrounding the unique zinc coordination site in lipase family I.5

“…Moreover, Timucin et al . has investigated the catalytic process of BTL2 was independent on zinc presence at ambient temperatures . Hence, these observations suggest that zinc in BTL2 possess structural roles in BTL2 without interfering with the catalytic action.…”

“…Zinc ions have been shown to activate or inhibit lipases by changing solubility of substrates and/or enzyme, or in catalytic properties of enzyme . The X‐ray structures of BTL2 (PDB ID: 2W22) and its W61A mutant (PDB ID: 5CE5) show that zinc ion is separated from the catalytic serine (S114) by more than 19 Å (Supporting Information Fig.…”

Effect of Site‐Specific Peptide‐Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus

“…This equilibrium can be completely shifted to obtain exclusively monomer molecules of lipase under correct conditions . Recently, it was shown that the addition or removal of zinc had an influence on the formation of enzyme aggregates or monomers, respectively …”