Switching the substrate specificity of lysoplasmalogen‐specific phospholipase D

Autor: Kazutaka Murayama, Takayuki Oyama, Daisuke Sugimori
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Models
Molecular
0301 basic medicine
substrate‐specificity switch
LysoPAF‐specific PLD
General Biochemistry
Genetics and Molecular Biology
Substrate Specificity
Structure-Activity Relationship
03 medical and health sciences
0302 clinical medicine
Phospholipase A2
Thermocrispum sp
phospholipase D
lysoplasmalogen‐specific PLD
Amino Acid Sequence
lcsh:QH301-705.5
Research Articles
Glycerophosphodiester phosphodiesterase
chemistry.chemical_classification
Molecular Structure
biology
Phosphoric Diester Hydrolases
Phospholipase D
Chemistry
lipoprotein‐associated phospholipase A2
Hydrolysis
Lipoprotein-associated phospholipase A2
Mutagenesis
Substrate (chemistry)
Recombinant Proteins
Actinobacteria
Kinetics
030104 developmental biology
Enzyme
Biochemistry
lcsh:Biology (General)
030220 oncology & carcinogenesis
Mutagenesis
Site-Directed
biology.protein
Specific activity
lipids (amino acids
peptides
and proteins)
Lysophospholipids
Research Article
Zdroj: FEBS Open Bio, Vol 11, Iss 4, Pp 1132-1143 (2021)
FEBS Open Bio
ISSN: 2211-5463
Popis: Lysoplasmalogen‐specific phospholipase D (LyPls‐PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well‐known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls‐PLD from Thermocrispum sp. RD004668 shows only 53% activity with 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) activity can be used to evaluate for cardiovascular disease. Hence, development of a point‐of‐care testing kit requires a LysoPAF‐specific PLD (LysoPAF‐PLD) to measure Lp‐PLA2 activity. Rational site‐directed mutagenesis and kinetic analysis were applied to generate LysoPAF‐PLD from LyPls‐PLD and to clarify the mechanisms underlying the substrate‐recognition ability of LyPls‐PLD. Our results suggest that LyPls‐PLD variants A47, M71, N173, F211, and W282 are possibly involved in substrate recognition and that F211L may substantially alter substrate preference. Moreover, the specific activity ratio LysoPAF/LyPlsCho corresponding to F211L was up to 25‐fold higher than that corresponding to the wild‐type enzyme. Thus, we succeeded in switching from LyPlsCho‐ to LysoPAF‐PLD. These results suggest that the F211L variant may be utilized to measure Lp‐PLA2 activity. Kinetic analyses demonstrated that product release was the rate‐limiting step of the reaction, with flexibility of the sn‐1 ether‐linked vinyl/alkyl chain of the substrate being essential for substrate binding and product release. Our findings may lead to a better understanding of the differences between homologous enzymes (such as PLD, sphingomyelinase D, and GDPD of the phosphatidylinositol‐phosphodiesterase superfamily) in relation to substrate recognition. Enzyme EC 3.1.4.2 (currently assigned).
Lysoplasmalogen‐specific phospholipase D (LyPls‐PLD) intrinsically has a high affinity for choline lysoplasmalogen (LyPlsCho). By rational site‐directed mutagenesis (F211L), we converted LysPls‐PLD to a version (LysoPAF‐PLD) that has a high affinity for LysoPAF rather than the natural substrate LyPlsCho. This F211L variant of LyPls‐PLD exhibited a complete shift in substrate specificity with no decrease in catalytic efficiency or turnover number.
Databáze: OpenAIRE
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