skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Thursday, May 23 until 2:00 AM ET on Friday, May 24 due to maintenance. We apologize for the inconvenience.


Title: A multidomain enzyme, with glycerol‐3‐phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii
Summary

Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green algaC. reinhardtiishowed substantial triacylglycerol (TAG) accumulation and up‐regulation of a gene,GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol‐3‐phosphate dehydrogenase (GPD) domains. CanonicalGPDenzymes catalyze the synthesis of glycerol‐3‐phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone ofTAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. RecombinantChlamydomonasGPD2 showed both reductase and phosphatase activitiesin vitroand it can work as a bifunctional enzyme capable of synthesizing glycerol directly fromDHAP. In addition,GPD2and a gene encoding glycerol kinase were up‐regulated inChlamydomonascells exposed to high salinity.RNA‐mediated silencing ofGPD2revealed that the multidomain enzyme was required forTAGaccumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, aGPD2‐mCherry fusion protein was found to localize to the chloroplast, supporting the existence of aGPD2‐dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities ofPSPGPDmultidomain enzymes may be modulated by post‐translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

 
more » « less
NSF-PAR ID:
10034635
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
The Plant Journal
Volume:
90
Issue:
6
ISSN:
0960-7412
Page Range / eLocation ID:
p. 1079-1092
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Summary

    Euonymus alatusdiacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to thesn‐3 position of diacylglycerol to form 3‐acetyl‐1,2‐diacyl‐sn‐glycerol (acetyl‐TAG).EaDAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed thatEaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology ofEaDAcT, we obtained an experimentally determined topology model for a plantMBOAT. TheEaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of theER. In addition, there is a large cytoplasmic loop between the first and secondTMDs, with theMBOATsignature region of the protein embedded in the thirdTMDclose to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase fromEuonymus fortuneipossessed the highest activityin vivoandin vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential forEaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity ofEaDAcT, suggesting that multiple amino acids are important for substrate recognition.

     
    more » « less
  2. Summary

    In the natural pesticides known as pyrethrins, which are esters produced in flowers ofTanacetum cinerariifolium(Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid.

    We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues.

    Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10‐carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of theSABATHmethyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries.

    Expression inN. benthamianaplants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

     
    more » « less
  3. Summary

    In plant lipid metabolism, the synthesis of many intermediates or end products often appears overdetermined with multiple synthesis pathways acting in parallel. Lipid metabolism is also dynamic with interorganelle transport, turnover, and remodeling of lipids. To explore this complexityin vivo, we developed anin vivolipid ‘tag and track’ method. Essentially, we probed the lipid metabolism inArabidopsis thalianaby expressing a coding sequence for a fatty acid desaturase fromPhyscomitrella patens(Δ6D). This enzyme places a double bond after the 6th carbon from the carboxyl end of an acyl group attached to phosphatidylcholine at itssn‐2 glyceryl position providing a subtle, but easily trackable modification of the glycerolipid. Phosphatidylcholine is a central intermediate in plant lipid metabolism as it is modified and converted to precursors for other lipids throughout the plant cell. Taking advantage of the exclusive location of Δ6D in the endoplasmic reticulum (ER) and its known substrate specificity for one of the two acyl groups on phosphatidylcholine, we were able to ‘tag and track’ the distribution of lipids within multiple compartments and their remodeling in transgenic lines of different genetic backgrounds. Key findings were the presence ofER‐derived precursors in plastid phosphatidylglycerol and prevalent acyl editing of thylakoid lipids derived from multiple pathways. We expect that this ‘tag and track’ method will serve as a tool to address several unresolved aspects of plant lipid metabolism, such as the nature and interaction of different subcellular glycerolipid pools during plant development or in response to adverse conditions.

     
    more » « less
  4. Summary

    Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition ofPATare currently unknown. Here we present the X‐ray crystal structures of the wild‐type and various mutants ofPATfromArabidopsis thaliana(AtPAT). Steady‐state kinetic and ligand‐binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution ofPATactivity among class IbAATenzymes. Structures of AtPATmutants co‐crystallized with either α‐ketoglutarate or pyridoxamine 5′‐phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor ofPATfromA. thaliana and Antirrhinum majusplants as well as the bacteriumChlorobium tepidum, uncovering a potential new effector ofPAT.

     
    more » « less
  5. Summary

    The GreenCut encompasses a suite of nucleus‐encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non‐photosynthetic/heterotrophic organisms. InChlamydomonas reinhardtii,CPLD49 (Conserved inPlantLineage andDiatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that acpld49mutant has impaired photoautotrophic growth under high‐light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochromeb6fcomplex (Cytb6f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore,CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein,CPLD38; a mutant null forCPLD38 also impacts Cytb6fcomplex accumulation. We investigated several potential functions ofCPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis thatCPLD38 andCPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6fcomplex. Based on motifs ofCPLD49 and the activities of otherCPLD49‐like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6f.

     
    more » « less