Revealing the Roles of Subdomains in the Catalytic Behavior of Lipases/Acyltransferases Homologous to CpLIP2 through Rational Design of Chimeric Enzymes

Abstract

The lipases/acyltransferases homologous to CpLIP2 of Candida parapsilosis efficiently catalyze acyltransfer reactions in lipid/water media with high water activity (a >0.9). Two new enzymes of this family, CduLAc from Candida dubliniensis and CalLAc8 from Candida albicans, were characterized. Despite 82 % sequence identity, the two enzymes have significant differences in their catalytic behaviors. In order to understand the roles played by the different subdomains of these proteins (main core, cap and C-terminal flap), chimeric enzymes were designed by rational exchange of cap and C-terminal flap, between CduLAc and CalLAc8. The results show that the cap region plays a significant role in substrate specificity; the main core was found to be the most important part of the protein for acyltransfer ability. Similar exchanges were made with CAL-A from Candida antarctica, but only the C-terminal exchange was successful. Yet, the role of this domain was not clearly elucidated, other than that it is essential for activity.

The modular structure of α/β‐hydrolases

“…This analysis was extended to the families of haloalkane dehalogenases and prolyl iminopeptidases, which are homologous to EH despite their different catalytic activities . In the meantime, additional domains were identified in other hydrolases, and their functional role was discussed, such as the N‐terminal β‐propeller domain in acyl aminoacyl peptidases , the role of subdomains in the catalytic behaviour of lipases and acyltransferases , the stabilizing N‐terminal domain in the murine liver EH (MLEH) or the importance of the C‐terminal domain in colipase binding of pancreatic lipases .…”

A Novel High-Throughput Assay Enables the Direct Identification of Acyltransferases

“…Finally, we tested whether previously identified acyltransferases also show activity in the newly developed assay. Together with the CAL-A-related acyltransferases originating from yeasts [4] and the acyltransferase from Mycobacterium smegmatis (MsAcT) [19], the esterases from Bacillus stearothermophilus (BsteE) [20] and Pseudomonas fluorescens (PFEQ) [21] were tested as controls. Besides Est8, MsAcT also shows detectable formation of oligocarbonates, revealing that the assay is not limited to detecting the acyltransferase activity of Est8 (Figure 4b).…”

“…The few currently known acyltransferases were identified from a pool of known hydrolases, as homologs thereof or discovered by accident [6][7][8]. Direct screening for novel acyltransferases from large libraries, e.g., from metagenome libraries, is unfeasible as typical acyltransferase assays are laborious and time-consuming, and thereby limit throughput [4,9]. If a collection of pre-selected hydrolases is screened to reduce library size, the identified acyltransferases will by definition have substantial hydrolase activity.…”

Characterization and Reshaping of a Large and Hydrophobic Nucleophile Pocket in Lipases/Acyltransferases

“…Herein, we report on investigations to better understand structure/function relationships in the lipase/acyltransferase groups.C haracteristics of already described enzymes ando f new singlea nd multiple mutants were collected and gathered in the classification previously established by Subileau et al,[2d] to propose am ost complete overview and comparison of actualk nowledgeo nl ipases/acyltransferases. One enzyme from each class was chosen as ar eference to design new targets for mutations:I )CduLAc from Candida dubliniensis, [5] II) C-pLIP2, and III)CAL-A. [2d] CAL-A shares 35 %s equence identity with CduLAc and3 2% with CpLIP2, whereasC duLAca nd CpLIP2 exhibit 60 %i dentity in their primary sequences.…”

“…Their sequences and 3D structures were aligned and comparedw ith the UCSF Chimerap ackage, [6] to highlight differences in the vicinity of the active site. As shown in Figure 1, the 3D models of wild-type CpLIP2, [7] CduLAc, [5] and the structure of CAL-A (PDB ID:2 VEO) [3a] exhibited ac avity that extended below the substrate-binding pocket and included the catalytic serine. In CpLIP2 and CduLAc,t his cavity is larger and more hydrophobic than that in CAL-A.W ehypothesized that this could contribute to the acyl transfer activity by providingamore favorable environment for alcohols than that for water,a nd may correspond to, or contain, the nucleophile site proposed by Rauwerdink and Kazlauskas, [8] in whichh ydrogenb ondingc an favor either hydrolysis or transfer reactions.…”