Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis

ABSTRACT The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.

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Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Life ◽

10.3390/life10100234 ◽

2020 ◽

Vol 10 (10) ◽

pp. 234

Author(s):

Zeineb Baklouti ◽

Cédric Delattre ◽

Guillaume Pierre ◽

Christine Gardarin ◽

Slim Abdelkafi ◽

...

Keyword(s):

Biochemical Characterization ◽

Biochemical Properties ◽

Cell Wall Degrading Enzymes ◽

Promising Candidate ◽

Structural Modifications ◽

Chimeric Enzymes ◽

Bifunctional Enzymes ◽

Trichoderma Sp ◽

Ph Variations

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

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Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.270.38.22399 ◽

1995 ◽

Vol 270 (38) ◽

pp. 22399-22405 ◽

Cited By ~ 108

Author(s):

Luc Berthiaume ◽

Marilyn D. Resh

Keyword(s):

Biochemical Characterization ◽

Acyltransferase Activity ◽

Palmitoyl Acyltransferase

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Triacylglycerol and phytyl ester synthesis inSynechocystissp. PCC6803

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915930117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 6216-6222 ◽

Cited By ~ 6

Author(s):

Mohammed Aizouq ◽

Helga Peisker ◽

Katharina Gutbrod ◽

Michael Melzer ◽

Georg Hölzl ◽

...

Keyword(s):

Fatty Acids ◽

Acyl Carrier Protein ◽

Sequence Similarity ◽

Wax Esters ◽

Oxygenic Photosynthesis ◽

Wax Ester ◽

Ester Synthesis ◽

Acyltransferase Activity ◽

Storage Lipids ◽

Triacylglycerol Synthesis

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacteriumSynechocystissp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis inSynechocystis. The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the geneslr2103responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

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Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

Applied and Environmental Microbiology ◽

10.1128/aem.03433-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 1132-1141 ◽

Cited By ~ 15

Author(s):

Juan A. Villa ◽

Matilde Cabezas ◽

Fernando de la Cruz ◽

Gabriel Moncalián

Keyword(s):

Neutral Lipids ◽

Limited Proteolysis ◽

Diacylglycerol Acyltransferase ◽

Wax Ester ◽

Sequence Motifs ◽

Acyltransferase Activity ◽

Structural Prediction ◽

Active Complex ◽

Acyl Coenzyme A ◽

Wax Ester Synthase

ABSTRACTTriacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.

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Identification and Characterization of a Novel Carboxylesterase EstQ7 from a Soil Metagenomic Library

10.21203/rs.3.rs-355216/v1 ◽

2021 ◽

Author(s):

Zhenzhen Yan ◽

Liping Ding ◽

Dandan Zou ◽

Luyao Wang ◽

Yuzhi Tan ◽

...

Keyword(s):

Biochemical Characterization ◽

Metagenomic Library ◽

Catalytic Triad ◽

Recombinant Enzyme ◽

Fatty Acid Esters ◽

Dimensional Structure ◽

Sequence Alignments ◽

Acyltransferase Activity ◽

Lipolytic Enzymes

Abstract A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174–Asp306–His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910 S-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Homology modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking reveled the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property, and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

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Characterization of a Soil Metagenome-Derived Gene Encoding Wax Ester Synthase

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1507.07029 ◽

2016 ◽

Vol 26 (2) ◽

pp. 248-254 ◽

Cited By ~ 5

Author(s):

Nam Hee Kim ◽

Ji-Hye Park ◽

Eunsook Chung ◽

Hyun-Ah So ◽

Myung Hwan Lee ◽

...

Keyword(s):

Wax Ester ◽

Gene Encoding ◽

Soil Metagenome ◽

Wax Ester Synthase

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Plant Pathology Diagnosed by X-Ray Microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129152 ◽

1987 ◽

Vol 45 ◽

pp. 974-975

Author(s):

J. H. Resau ◽

N. Howell ◽

S. H. Chang

Keyword(s):

Electron Microscopy ◽

Plant Pathology ◽

Biochemical Characterization ◽

Nutrient Deficiency ◽

Green Leaf ◽

Parasitic Infestation ◽

X Ray

Spinach grown in Texas developed “yellow spotting” on the peripheral portions of the leaves. The exact cause of the discoloration could not be determined as there was no evidence of viral or parasitic infestation of the plants and biochemical characterization of the plants did not indicate any significant differences between the yellow and green leaf portions of the spinach. The present study was undertaken using electron microscopy (EM) to determine if a micro-nutrient deficiency was the cause for the discoloration.Green leaf spinach was collected from the field and sent by express mail to the EM laboratory. The yellow and equivalent green portions of the leaves were isolated and dried in a Denton evaporator at 10-5 Torr for 24 hrs. The leaf specimens were then examined using a JEOL 100 CX analytical microscope. TEM specimens were prepared according to the methods of Trump et al.

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