Mutagenesis and homology modeling reveal a predicted pocket of lysophosphatidylcholine acyltransferase 2 to catch Acyl‐CoA

Hamano, Fumie; Matoba, Kazuaki; Hashidate-Yoshida, Tomomi; Suzuki, Tomoyuki; Miura, Kiyotake; Hishikawa, Daisuke; Harayama, Takeshi; Yuki, Koichi; Kita, Yoshiaki; Noda, Nobuo N.; Shimizu, Takao; Shindou, Hideo

doi:10.1096/fj.202002591r

Cited by 4 publications

(9 citation statements)

References 37 publications

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“…As previously reported, PlsC proteins are characterized by four conserved motifs that are proposed to surround a hydrophobic pocket containing the active site (motifs I-IV; Fig. 2A) [21,23]. Comparison between the amino acid sequences of E. coli PlsC and Xanthomonas PlsC homologs revealed the presence of at least two regions (motif I and motif III) with a high degree of conservation.…”

Section: The Importance Of Conserved Motifs In Xc_plsc2 and Xc_plsc3supporting

confidence: 66%

“…The conserved amino acids in the acyltransferase motifs I–IV are characteristic for PlsCs [21], and many of the amino acids, particularly the arginine residues from motifs II and III, have been shown to be crucial for LPA to PA acylation [22,23]. In Xc_0505, both motifs II and III differ substantially from the consensus sequence and lack the essential arginines (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PlsC enzymes are characterized by four conserved motifs (motifs I-IV; Fig. 2A) [21], which are important for catalytic activity [22,23]. BLAST analyses with the two LPAAT from E. coli (PlsC and YihG) as queries suggested five PlsC candidates in X. campestris (Xc_4099, Xc_0188, Xc_0238, Xc_0517, and Xc_0505).…”

Section: Xanthomonas Campestris Encodes Multiple Putative Plsc Homologsmentioning

confidence: 99%

“…Motif I (HxxxxD) is part of the active site and is highly conserved in various acyltransferases from eukaryotic and prokaryotic organisms [22,23]. In Xc_PlsC3, mutation of the histidine at position 65 to alanine (H65A) abolished the ability to rescue the temperature-sensitive growth of the E. coli plsC mutant (Fig.…”

Section: The Importance Of Conserved Motifs In Xc_plsc2 and Xc_plsc3mentioning

confidence: 99%

“…In contrast to other PlsCs, point mutations of the arginines in Xc_PlsC2 motif III, which is proposed to be critical for anchoring the phosphate of LPA [17,22], did not affect complementation activity. While the canonical arginine is an essential part of the active site of LPAATs [17,22], this residue is not part of motif III of the mouse LPC acyltransferase mLPCAT2 [23]. The nonessential role and unusual order of residues in motif III of Xc_PlsC2, in concert with its previously reported function as an LPC acyltransferase [18] suggest that the enzyme is not a strict LPAAT, but perhaps an acyltransferase with a broad range of accepted substrates as reported for R. capsulatus OlsA [12] and E. coli YihG [14].…”

Section: Are All Four Plsc Homologs Involved In Lpa To Pa Acylation I...mentioning

confidence: 99%

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Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris

Vasilopoulos,

Heflik,

Czolkoss

et al. 2023

The FEBS Journal

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Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol‐3‐phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC‐like activity was supported by the functional complementation of a temperature‐sensitive plsC‐deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC‐like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent‐solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

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Section: The Importance Of Conserved Motifs In Xc_plsc2 and Xc_plsc3supporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Xanthomonas Campestris Encodes Multiple Putative Plsc Homologsmentioning

confidence: 99%

Section: The Importance Of Conserved Motifs In Xc_plsc2 and Xc_plsc3mentioning

confidence: 99%

Section: Are All Four Plsc Homologs Involved In Lpa To Pa Acylation I...mentioning

confidence: 99%

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Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris

Vasilopoulos,

Heflik,

Czolkoss

et al. 2023

The FEBS Journal

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Update and nomenclature proposal for mammalian lysophospholipid acyltransferases, which create membrane phospholipid diversity

Valentine

Yanagida

Kawana

et al. 2022

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification of a 1‐acyl‐glycerol‐3‐phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis

Santoshi,

Bansia,

Hussain

et al. 2024

Molecular Microbiology

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Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol‐3‐phosphate, wherein the second acylation step is catalyzed by acylglycerol‐3‐phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane‐bound enzyme, and functions as 1‐acylglycerol‐3‐phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol‐3‐phosphate (LPA) from monounsaturated fatty acyl‐coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

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Mutagenesis and homology modeling reveal a predicted pocket of lysophosphatidylcholine acyltransferase 2 to catch Acyl‐CoA

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 4 publications

References 37 publications

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris

Update and nomenclature proposal for mammalian lysophospholipid acyltransferases, which create membrane phospholipid diversity

Identification of a 1‐acyl‐glycerol‐3‐phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis

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