Schizochytrium sp., a eukaryotic microalga, biosynthesizes docosahexaenoic acid (DHA; C22:6 ω3) by DHA synthase, which is composed of three subunits (OrfA to C) possessing multiple catalytic domains. The enzyme has three dehydratase (DH) domains, DHPKS in OrfA and tandem DHFabA (DH1FabA and DH2FabA) domains in OrfC. In this study, the function of each of the DH domains was investigated by in vivo heterologous expression experiments in Escherichia coli and in vitro studies with recombinant truncated OrfA containing DHPKS and full-length OrfC, in which one of the tandem DHFabA domains was inactivated by site-directed mutagenesis, and acyl–acyl carrier protein (ACP) intermediates. Following heterologous expression, the DH1FabA- or DH2FabA-inactivated enzyme produced no DHA, revealing that both domains are essential, while the DHPKS-inactivated enzyme showed significantly reduced DHA productivity, confirming that DHPKS is required for full production of DHA. Moreover, in vitro experiments showed that the C4-ACP substrate was accepted by all of the DHs, but DHPKS and DH2FabA showed higher activities than DH1FabA. DH1FabA catalyzed two types of reactions: (de)hydrations of C10-, C16-, and C22-ACP substrates and (de)hydrations/2-trans to 2-cis isomerization of C8-, C14-, and C20-ACP substrates. By contrast, DH2FabA catalyzed (de)hydrations/2-trans to 3-cis isomerization of C6-, C12-, and C18-ACP substrates.
Schizochytrium sp.是一种真核微藻,通过DHA合成酶生物合成二十二碳六烯酸(DHA; C22:6 ω3),该合成酶由三个亚基(OrfA to C)组成,具有多个催化结构域。该酶具有三个脱水酶(DH)结构域,OrfA中的DHPKS和OrfC中的串联DHFabA (DH1FabA和DH2FabA)结构域。在本研究中,通过大肠杆菌的体内外源表达实验和含有DHPKS和全长OrfC的重组截断OrfA的体外研究,研究了每个DH结构域的功能,其中一个串联DHFabA结构域通过定点诱变和酰基酰基载体蛋白(ACP)中间体失活。异种表达后,DH1FabA-或dh2faba -失活酶不产生DHA,这表明这两个结构域都是必需的,而DHPKS失活酶的DHA产量显著降低,证实了DHPKS是完全产生DHA所必需的。此外,体外实验表明,C4-ACP底物被所有DHs接受,但DHPKS和DH2FabA的活性高于DH1FabA。DH1FabA催化两种类型的反应:C10-、C16-和C22-ACP底物的(脱)水合反应和C8-、C14-和C20-ACP底物的(脱)水合/2-反式到2-顺式异构化反应。相比之下,DH2FabA催化C6-、C12-和C18-ACP底物的(脱)水合/2-反式到3-顺式异构化。
{"title":"Functional Analysis of Three Dehydratase Domains, DHPKS, DH1FabA, and DH2FabA, in Microalgal Docosahexaenoic Acid Synthase","authors":"Tomoya Kawata, Hiyu Kobayashi, Makoto Otsuka, Chitose Maruyama, Yoshimitsu Hamano, Takeshi Tsunoda, Yasushi Ogasawara, Tohru Dairi","doi":"10.1002/ceur.202500312","DOIUrl":"https://doi.org/10.1002/ceur.202500312","url":null,"abstract":"<p><i>Schizochytrium</i> sp., a eukaryotic microalga, biosynthesizes docosahexaenoic acid (DHA; C22:6 <i>ω</i>3) by DHA synthase, which is composed of three subunits (OrfA to C) possessing multiple catalytic domains. The enzyme has three dehydratase (DH) domains, DH<sub>PKS</sub> in OrfA and tandem DH<sub>FabA</sub> (DH1<sub>FabA</sub> and DH2<sub>FabA</sub>) domains in OrfC. In this study, the function of each of the DH domains was investigated by in vivo heterologous expression experiments in <i>Escherichia coli</i> and in vitro studies with recombinant truncated OrfA containing DH<sub>PKS</sub> and full-length OrfC, in which one of the tandem DH<sub>FabA</sub> domains was inactivated by site-directed mutagenesis, and acyl–acyl carrier protein (ACP) intermediates. Following heterologous expression, the DH1<sub>FabA</sub>- or DH2<sub>FabA</sub>-inactivated enzyme produced no DHA, revealing that both domains are essential, while the DH<sub>PKS</sub>-inactivated enzyme showed significantly reduced DHA productivity, confirming that DH<sub>PKS</sub> is required for full production of DHA. Moreover, in vitro experiments showed that the C4-ACP substrate was accepted by all of the DHs, but DH<sub>PKS</sub> and DH2<sub>FabA</sub> showed higher activities than DH1<sub>FabA</sub>. DH1<sub>FabA</sub> catalyzed two types of reactions: (de)hydrations of C10-, C16-, and C22-ACP substrates and (de)hydrations/2-<i>trans</i> to 2-<i>cis</i> isomerization of C8-, C14-, and C20-ACP substrates. By contrast, DH2<sub>FabA</sub> catalyzed (de)hydrations/2-<i>trans</i> to 3-<i>cis</i> isomerization of C6-, C12-, and C18-ACP substrates.</p>","PeriodicalId":100234,"journal":{"name":"ChemistryEurope","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/ceur.202500312","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145904650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Front Cover shows a porosity-controlled carbon fiber paper, constructed by electrospinning polyacrylonitrile (PAN) and poly(methyl 2-methacrylate) (PMMA) and carbonization, as a dual-functional electrode substrate for high-sulfur-loading cathodes (6.1 mg cm−2) and stabilized lean-lithium anodes (10 mA h cm−2). The fabricated lithium–sulfur full cell achieves stable 400 cycles with a low negative-to-positive capacity ratio under lean electrolyte conditions (electrolyte-to-sulfur ratio of 7 μL mg−1). For more information, see the Research Article by C.-C. Wu and S.-H. Chung (DOI: 10.1002/ceur.202500070).