Molecular cloning, heterologous expression, and enzymatic characterization of lysoplasmalogen‐specific phospholipase D from <i>Thermocrispum</i> sp.

<jats:sec><jats:label/><jats:p>Lysoplasmalogen (LyPls)‐specific phospholipase D (LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content>) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1‐(1‐alkenyl)‐<jats:italic>sn</jats:italic>‐glycero‐3‐phosphate (lysoplasmenic acid). Little is known about LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> 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‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> from <jats:italic>Thermocrispum</jats:italic> sp. strain <jats:styled-content style="fixed-case">RD</jats:styled-content>004668, and the enzyme was purified, characterized, cloned, and expressed using <jats:styled-content style="fixed-case">pET</jats:styled-content>24a(+)/<jats:italic>Escherichia coli</jats:italic> with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (<jats:styled-content style="fixed-case">pH</jats:styled-content> 8.0 and 50 °C), steady‐state kinetic analysis for LyPlsCho yielded <jats:italic>K</jats:italic><jats:sub>m</jats:sub> and <jats:italic>k</jats:italic><jats:sub>cat</jats:sub> values of 13.2 μ<jats:sc>m</jats:sc> and 70.6 s<jats:sup>−1</jats:sup>, respectively. The <jats:styled-content style="fixed-case">ORF</jats:styled-content> of LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> gene consisted of 1005 bp coding a 334‐amino‐acid (aa) protein. The deduced aa sequence of LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> showed high similarity to those of glycerophosphodiester phosphodiesterases (<jats:styled-content style="fixed-case">GDPD</jats:styled-content>s); however, the substrate specificity differed completely from those of <jats:styled-content style="fixed-case">GDPD</jats:styled-content>s and general phospholipase Ds (<jats:styled-content style="fixed-case">PLD</jats:styled-content>s). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> were highly conserved to <jats:styled-content style="fixed-case">GDPD</jats:styled-content>s. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content> may participate in the substrate recognition. These findings will help to elucidate differences among LyPls‐<jats:styled-content style="fixed-case">PLD</jats:styled-content>,<jats:styled-content style="fixed-case"> PLD</jats:styled-content>, and <jats:styled-content style="fixed-case">GDPD</jats:styled-content> with regard to function, substrate recognition mechanism, and biochemical roles.</jats:p></jats:sec><jats:sec><jats:title>Data Accessibility</jats:title><jats:p><jats:italic>Thermocrispum</jats:italic> sp. 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.</jats:p></jats:sec><jats:sec><jats:title>Enzyme</jats:title><jats:p>EC number <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/4/4.html">EC 3.1.4.4</jats:ext-link></jats:p></jats:sec>