Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen-specific phospholipase D fromThermocrispumsp.
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 fromThermocrispumsp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coliwith 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 °C), steady-state kinetic analysis for LyPlsCho yieldedKmandkcatvalues of 13.2 μm 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.
Thermocrispumsp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP-01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls-PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively.
EC number EC 3.1.4.4.
Department of Symbiotic Systems Science and Technology Graduate School of Symbiotic Systems Science and Technology Fukushima University Japan.
Yusaku
Y
Matsumoto
Department of Symbiotic Systems Science and Technology Graduate School of Symbiotic Systems Science and Technology Fukushima University Japan.
Nana
N
Kashiwabara
Department of Symbiotic Systems Science and Technology Graduate School of Symbiotic Systems Science and Technology Fukushima University Japan.
Takayuki
T
Oyama
Division of Biomedical Measurements and Diagnostics Graduate School of Biomedical Engineering Tohoku University Sendai Japan.
Kazutaka
K
Murayama
Asahi Kasei Pharma Corp. Izunokuni Shizuoka Japan.
Hideyuki
H
Matsumoto
Asahi Kasei Pharma Corp. Izunokuni Shizuoka Japan.
Shin-Ich
SI
Sakasegawa
Department of Symbiotic Systems Science and Technology Graduate School of Symbiotic Systems Science and Technology Fukushima University Japan.
Daisuke
D
Sugimori
Thermocrispum sp.; biochemical characterization; cloning and heterologous expression; head group recognition; lysoplasmalogen‐specific phospholipase D