Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D from Thermocrispum sp.

作者：Matsumoto Yusaku; Kashiwabara Nana; Oyama Takayuki; Murayama Kazutaka; Matsumoto Hideyuki; Sakasegawa Shin ich; Sugimori Daisuke

来源：FEBS Open Bio, 2016, 6(11): 1113-1130.

DOI：10.1002/2211-5463.12131

• biochemical characterization
• cloning and heterologous expression
• head group recognition
• lysoplasmalogen-specific phospholipase D
• Thermocrispum sp.

Lysoplasmalogen (LyPls)-specific phospholipase D (LyPls-PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1-(1-alkenyl)-sn-glycero-3-phosphate (lysoplasmenic acid). Little is known about LyPls-PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls-PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (pH 8.0 and 50 degrees C), steady-state kinetic analysis for LyPlsCho yielded K-m and k(cat) values of 13.2 m and 70.6 s(-1), respectively. The ORF of LyPls-PLD gene consisted of 1005 bp coding a 334-amino-acid (aa) protein. The deduced aa sequence of LyPls-PLD showed high similarity to those of glycerophosphodiester phosphodiesterases (GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds (PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls-PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls-PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls-PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles.

• 出版日期2016-11