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.
Highlights
The understanding of sequence-encoded information has never been as important as in this post-genomic era where protein purification and characterization is dramatically outpaced by the volume of DNA sequences deposited in various databanks
We previously suggested that the novel Geobacillus thermodenitrificans EstGtA2 diverged from the classification proposed by Arpigny and Jaeger, and was instead a representative of a new family of thermostable lipolytic enzymes named N’ [7]
We proposed the 15th family which correspond to abH11.03 on the basis of a distinct phylogenetic cluster (< 32% with family XIII), (Figure S8), the architecture of the cap domain (α/β cap) compared with the three α-helices cap of Est30-like proteins and suggest its separation and identification of a subfamily named N’ on the basis of a distinctive salt bridges pattern and on the polarity of a conserved interloop salt bridge in the active site
Summary
The understanding of sequence-encoded information has never been as important as in this post-genomic era where protein purification and characterization is dramatically outpaced by the volume of DNA sequences deposited in various databanks. Carboxylesterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) are lipolytic enzymes belonging to the class of hydrolases which catalyze the hydrolysis or synthesis of ester bonds in lipids. They adopt the α/β hydrolase fold and bear a catalytic triad formed by Ser-Asp/Glu-His, but differ in term of substrate specificity [1,2]. Prokaryotic lipolytic enzymes have been assigned to eight different families (I-VIII) based on sequence identity and biochemical properties by Arpigny and Jaeger [3]. Despite important similarities between N’ and Est30-like enzymes (family XIII), the N’-related enzymes form a clearly distinct group including monoacylglycerol lipases. We will revisit the various classifications or family names suggested for these lipolytic enzymes and attempt to reconcile them by proposing their assignment to the new 15th family (family XV), named chronologically based on the extension of the Arpigny and Jaeger’s classification
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