Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

Abstract

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/ T560W was generated, which had the highest catalytic efficiency (k cat /K m ) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

“…The multiple sequence alignment for protein structure prediction was obtained by the HHPred Server [25] and compared with the results of threading methods, as Phyre [26] and GeneSilico Metaserver [27]. The optimization of the multiple alignments for 3D modeling was carried out by hand, according to information on functional and conserved residues, and secondary structures.…”

Neurotoxins and Their Binding Areas on Voltage-Gated Sodium Channels

“…The revised binding site model can specifically signify a huge simplification of the conventional site 3 and site 4 since their current pharmacological profiles become more and more complex (Billen et al, 2010; Bosmans and Swartz, 2010; Liu et al, 2011). In general, the location of the site 4 binding area in the revised model can remain identical to the conventional site 4, located at DII, comprising the extracellular linkers between S1–S3 and S3–S4.…”

“…This can explain the apparent paradox as is the case for site 3 toxins like BmK I and BmK αIV (α-like scorpion toxins from B. martensii Karsch) that can displace radiolabeled site 4 toxins like BmK AS (β-like scorpion toxin) and BmK IT2 (depressant insect β-scorpion toxin) respectively (Li et al, 2000; Chai et al, 2006). For a comprehensive review on BmK toxins and another binding model for the complex sites 3 and 4 (see Billen and Tytgat, 2009; Liu et al, 2011). …”

Biocatalyst development by directed evolution

“…A variant (R526E) was identified by saturation mutagenesis on a conserved residue in the active site, which essentially switched this peptidase into an esterase (Wang et al, 2006). Further mutagenesis by error-prone PCR and saturation mutagenesis revealed a variant with up to 280-fold increased esterase activity on a more bulky substrate while maintaining high thermostability (Liu et al, 2011a). In another example, the thermostable lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes neurotoxic organophosphorus compounds at a lower rate than its natural substrate lactones.…”