Role of Key Salt Bridges in Thermostability of G. thermodenitrificans EstGtA2: Distinctive Patterns within the New Bacterial Lipolytic Enzyme Family XV

Abstract

Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15th family (family XV) and suggest criteria for the assignation of protein sequences to the N’ subfamily. Five selected salt bridges, hallmarks of the N’ subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N’ subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.

An insight into microbial lipases and their environmental facet

“…The presence of charged residues and the formation of salt bridges play a key role in thermostability. The accumulation of more than two mutations had a dramatic impact on Geobacillus thermodenitrificans EstGtA2 lipase activity at high temperatures, suggesting an important role of conserved salt bridge-forming residues in thermostability (Charbonneau and Beauregard, 2013). Alkaline lipases are widely produced by the members of Bacillus genera, including B. licheniformis (Chakraborty and Raj 2008).…”

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

“…Nevertheless, we clearly characterized RCM Bmas as a mesophilic enzyme showing a temperature optimum of 35°C, while RCM Ktus has its activity maximum at 55°C (12). This thermal adaptation may be caused by various molecular mechanisms, such as the number of salt bridges and hydrophobic interactions (27). In particular, the flexibility of surface-exposed loops may be crucial, as has been recently demonstrated for esterases with conserved biochemical properties but substantially different temperature optima (28).…”

Characterization of an alkaline esterase from an enriched metagenomic library derived from an oil-spill area

“…Bacterial lipolytic enzymes were originally classified into eight families based their sequences and biological properties [4], and expanded to fifteen families [5]. Besides microorganisms, biocatalysts have been explored using a metagenomic approach without culturing of microorganisms, due to the limitation of culture [6].…”