Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering
| Autor: | Jasmina Damnjanović, Hideo Nakano, Yugo Iwasaki |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Stereochemistry
Mutation Missense Bioengineering 01 natural sciences Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Phospholipase D Saturated mutagenesis Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Liposome 010405 organic chemistry Chemistry Streptomyces antibioticus Substrate (chemistry) Lysophosphatidylcholines Phosphatidic acid Protein engineering 0104 chemical sciences Lysophosphatidylcholine Enzyme Amino Acid Substitution Mutagenesis Site-Directed lipids (amino acids peptides and proteins) Biotechnology |
| Zdroj: | Protein engineering, designselection : PEDS. 32(1) |
| ISSN: | 1741-0134 |
| Popis: | Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7–10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes. |
| Databáze: | OpenAIRE |
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