Structural and biochemical characterization of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 reveal its stepwise catalytic mechanism

Liu, Yinghui; Feng, Yanbin; Wang, Yayue; Li, Xia; Cao, Xupeng; Xue, Song

doi:10.1016/j.bbrc.2015.01.003

Cited by 6 publications

(4 citation statements)

References 29 publications

(23 reference statements)

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“…Cells with higher Fv/Fm value imply the stable physiological status of the cell without being impaired by photoinhibition [ 25 ]. The previous studies have reported that provision of fatty acid precursors and protection of photosynthetic apparatus could enhance photosynthetic performance [ 2 , 26 , 27 ]. It is well known that AGPAT1 plays a significant role in producing phosphatidic acid (PA), a key fatty acid component in the synthesis of plastidial membrane, lipids etc.…”

Section: Resultsmentioning

confidence: 99%

Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum

Balamurugan

Wang

et al. 2017

Biotechnol Biofuels

123

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BackgroundMicroalgae have emerged as a potential feedstock for biofuels and bioactive components. However, lack of microalgal strains with promising triacylglycerol (TAG) content and desirable fatty acid composition have hindered its commercial feasibility. Attempts on lipid overproduction by metabolic engineering remain largely challenging in microalgae.ResultsIn this study, a microalgal 1-acyl-sn-glycerol-3-phosphate acyltransferase designated AGPAT1 was identified in the model diatom Phaeodactylum tricornutum. AGPAT1 contained four conserved acyltransferase motifs I–IV. Subcellular localization prediction and thereafter immuno-electron microscopy revealed the localization of AGPAT1 to plastid membranes. AGPAT1 overexpression significantly altered the primary metabolism, with increased total lipid content but decreased content of total carbohydrates and soluble proteins. Intriguingly, AGPAT1 overexpression coordinated the expression of other key genes such as DGAT2 and GPAT involved in TAG synthesis, and consequently increased TAG content by 1.81-fold with a significant increase in polyunsaturated fatty acids, particularly EPA and DHA. Moreover, besides increased lipid droplets in the cytosol, ultrastructural observation showed a number of TAG-rich plastoglobuli formed in plastids.ConclusionThe results suggested that AGPAT1 overexpression could elevate TAG biosynthesis and, moreover, revealed the occurrence of plastidial TAG synthesis in the diatom. Overall, our data provide a new insight into microalgal lipid metabolism and candidate target for metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0786-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum

Balamurugan

Wang

et al. 2017

Biotechnol Biofuels

123

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“…Interestingly, no conversion of acetoacetyl‐CoA and malonyl‐CoA is detected when the crystal is soaked with these substrates at pH 4.6, whereas at pH 5.5 the crystals deteriorate when soaked with acetoacetyl‐CoA or malonyl‐CoA. If the YDCF cysteine acts as a nucleophile, then the function of the enzyme could be a malonyl‐CoA transacylase, such as a malonyl‐CoA:ACP transacylase (MCAT), transferring the malonyl moiety from CoA to ACP via a covalent malonylated intermediate . Malonyl‐ACP is a substrate in fatty acid synthesis reactions.…”

Section: Discussionmentioning

confidence: 99%

The SCP2-thiolase-like protein (SLP) ofTrypanosoma bruceiis an enzyme involved in lipid metabolism

et al. 2016

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Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5-fold reduction of de novo sterol biosynthesis from glucose- and acetate-derived acetyl-CoA. Fluorescence analyses of EGFP-tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP-acetoacetyl-CoA (1.90 Å) and TbSLP-malonyl-CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl-CoA tightly (Kd 90 µM), acetoacetyl-CoA moderately (Kd 0.9 mM) and acetyl-CoA and CoA very weakly. TbSLP possesses low malonyl-CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075-1096. © 2016 Wiley Periodicals, Inc.

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“…Malonyl-ACP is challenging to prepare for multiple reasons, the first of which is that holo-ACP is required as a substrate (Fig 1) [11]. Holo-ACP is formed by the transfer of phosphopantetheine from CoA to serine 36 of apo-ACP, the unmodified polypeptide [12,13].…”

Section: Introductionmentioning

confidence: 99%

“…This reaction can be performed on the benchtop or in vivo with overexpression of the holo-ACP synthase gene [11]. Malonyl-ACP is then formed by the transfer of the malonyl moiety from malonyl-CoA to holo-ACP and is catalyzed by the malonly-CoA:ACP transacylase, FabD [14,15].…”

Section: Introductionmentioning

confidence: 99%

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development

Marcella

Jing

Barb

2015

Protein Expression and Purification

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The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays.

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Structural and biochemical characterization of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 reveal its stepwise catalytic mechanism

Cited by 6 publications

References 29 publications

Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum

Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum

The SCP2-thiolase-like protein (SLP) ofTrypanosoma bruceiis an enzyme involved in lipid metabolism

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development

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