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Violaxanthin de-epoxidase and its closest relative: identification and characterization

Guo, Kuo LU (2017)
Abstract (Swedish)
Växter behöver ljus för att växa. Ljuset omvandlas till kemisk energi genom fotosyntes i växtens blad. Den kemiska energin kan sedan användas av växten. Ljusabsorptionen i växter har en begränsning. Alltför mycket ljus kommer att orsaka bildning av högenergimolekyler som är skadliga för växterna. Därför måste växter justera mängden ljus som tas upp. Ljuset i miljön förändras ständigt och exponeringen för starkt ljus kan också variera med tiden. Växter har utvecklat olika lösningar på problemet med alltför starkt ljus i olika tidsskalor. Ett snabbt sätt för växter att omvandla överskott av ljusenergi till värme kräver ett pigment som kallas zeaxanthin. Zeaxanthin bildas från ett annat pigment som kallas violaxanthin med hjälp av ett enzym... (More)
Växter behöver ljus för att växa. Ljuset omvandlas till kemisk energi genom fotosyntes i växtens blad. Den kemiska energin kan sedan användas av växten. Ljusabsorptionen i växter har en begränsning. Alltför mycket ljus kommer att orsaka bildning av högenergimolekyler som är skadliga för växterna. Därför måste växter justera mängden ljus som tas upp. Ljuset i miljön förändras ständigt och exponeringen för starkt ljus kan också variera med tiden. Växter har utvecklat olika lösningar på problemet med alltför starkt ljus i olika tidsskalor. Ett snabbt sätt för växter att omvandla överskott av ljusenergi till värme kräver ett pigment som kallas zeaxanthin. Zeaxanthin bildas från ett annat pigment som kallas violaxanthin med hjälp av ett enzym som kallas violaxanthin de-epoxidas (VDE). Denna reaktion aktiveras när bladen är under lätt ljus-stress.

En del av detta arbete handlar om att karakterisera VDE. Funktion av ett protein är alltid kopplad till dess sekvens och struktur. Byggstenarna för att göra ett protein kallas aminosyror där varje aminosyra har olika egenskaper. Olika aminosyror kopplas ihop i en specifik ordning och ger proteinet dess egenskaper. En typ av aminosyror, cystein, har en speciell förmåga att bindas till varandra under oxidativt tillstånd. Sådana cystein-cystein bryggor har tidigare visat sig bidra till proteiners struktur och katalytiska funktion. I detta arbete har vi bytt ut varje cystein till en annan aminosyra som har liknande egenskaper men som inte kan bilda bryggor. På så sätt identifierade vi cysteinernas betydelse i VDE. Sekvensen av VDE har tre delar. I detta arbete separerade vi VDE i delar och försökte ta reda på vikten av varje del.

En annan del av detta arbete handlar om ett protein som är nära släkt med VDE. Detta protein kallas VDE-relaterat protein (VDR). En viktig likhet mellan de två proteinerna är att de har mycket liknande arrangemang av cysteiner i sin sekvens. Vi gjorde många ytterligare analyser för att försöka kartlägga skillnader och likheter mellan dessa två proteiner. Vi gjorde också en del analyser på VDR för att försöka identifiera dess egenskaper, men dess funktion är fortfarande inte klargjord.

Sammantaget låter dessa resultat oss förstå VDR bättre och samtidigt ökar vår kunskap om VDE, vilket i förlängningen kan användas för förståelse av växtens funktion och i växtförädling.
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Abstract
Light is essential for plants and algae to process photosynthesis. However, excess of light will cause damage to the organism. A process called non-photochemical quenching (NPQ) is an important way for these organisms to protect themselves from photo oxidative damage. The NPQ process is depend on the xanthophyll cycle in thylakoids, which is controlled by the Violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). VDE converts Violaxanthin to to Zeaxanthin on the lumen side of the thylakoid membrane under acid pH conditions caused by photosynthesis.

The mature VDE sequence can be divided into three domains, an N-terminal domain with conserved cysteine pattern, a lipocalin-like domain which expected to carry the substrate... (More)
Light is essential for plants and algae to process photosynthesis. However, excess of light will cause damage to the organism. A process called non-photochemical quenching (NPQ) is an important way for these organisms to protect themselves from photo oxidative damage. The NPQ process is depend on the xanthophyll cycle in thylakoids, which is controlled by the Violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). VDE converts Violaxanthin to to Zeaxanthin on the lumen side of the thylakoid membrane under acid pH conditions caused by photosynthesis.

The mature VDE sequence can be divided into three domains, an N-terminal domain with conserved cysteine pattern, a lipocalin-like domain which expected to carry the substrate binding site, and a C-terminal domain rich in glutamic acids. In this work, we have constructed cysteine mutants that revealed that 12 of the 13 cysteines in VDE are essential for the activity; instead of directly contribute to catalytic function, these 12 cysteines formed disultphide bonds for VDE structural folding.

The catalytic center of VDE does not appear to be only located in the cysteine-rich N-terminal domain. The expressed N-terminal domain did not show activity and the N-terminal truncation of VDE also loss catalytic ability. When mixing these two separately expressed peptides together, the activity was regained. This shows that these two domains could fold independently to their active folding, and also indicates that the active site may be located in the interface of the two domains.

The closest relative of VDE has been found through bioinformatics analysis, the protein has a conserved cysteine pattern as VDE, and named as VDE related protein (VDR). From bioinformatics analysis, some characterization of VDR has been developed; VDR and VDE could be found in same organisms in tree of life, and they are suggested to have the same ancestor; cysteine-rich domain between the two protein are expected to have similar spatial structure, while the corresponding domain of lipocalin-like domain in VDR could have different structure as VDE. With 5’ RACE method and western blot, mRNA and protein level evidence of VDR expression is confirmed, together with differential centrifugation, the localization of VDR in leaf would be suggested to follow chlorophyll.
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Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Spetea Wiklund, Cornelia, Department of Biological and Environmental Sciences, University of Gothenburg
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Photosynthesis, Carotenoid conversions, violaxanthin de-epoxidase (VDE), Violaxanthin de-epoxidase related protein
pages
112 pages
publisher
Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology
defense location
Lecture hall A, Center for chemistry and chemical engineering, Naturvetarvägen 14, Lund
defense date
2017-12-12 13:15
ISBN
978-91-7422-552-5
978-91-7422-551-8
language
English
LU publication?
yes
id
14e35d03-32a3-49e9-a550-4908e9e993aa
date added to LUP
2017-11-16 13:06:25
date last changed
2017-11-22 23:58:27
@phdthesis{14e35d03-32a3-49e9-a550-4908e9e993aa,
  abstract     = {Light is essential for plants and algae to process photosynthesis. However, excess of light will cause damage to the organism. A process called non-photochemical quenching (NPQ) is an important way for these organisms to protect themselves from photo oxidative damage. The NPQ process is depend on the xanthophyll cycle in thylakoids, which is controlled by the Violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). VDE converts Violaxanthin to to Zeaxanthin on the lumen side of the thylakoid membrane under acid pH conditions caused by photosynthesis. <br/><br/>The mature VDE sequence can be divided into three domains, an N-terminal domain with conserved cysteine pattern, a lipocalin-like domain which expected to carry the substrate binding site, and a C-terminal domain rich in glutamic acids. In this work, we have constructed cysteine mutants that revealed that 12 of the 13 cysteines in VDE are essential for the activity; instead of directly contribute to catalytic function, these 12 cysteines formed disultphide bonds for VDE structural folding. <br/><br/>The catalytic center of VDE does not appear to be only located in the cysteine-rich N-terminal domain. The expressed N-terminal domain did not show activity and the N-terminal truncation of VDE also loss catalytic ability. When mixing these two separately expressed peptides together, the activity was regained. This shows that these two domains could fold independently to their active folding, and also indicates that the active site may be located in the interface of the two domains.<br/><br/>The closest relative of VDE has been found through bioinformatics analysis, the protein has a conserved cysteine pattern as VDE, and named as VDE related protein (VDR). From bioinformatics analysis, some characterization of VDR has been developed; VDR and VDE could be found in same organisms in tree of life, and they are suggested to have the same ancestor; cysteine-rich domain between the two protein are expected to have similar spatial structure, while the corresponding domain of lipocalin-like domain in VDR could have different structure as VDE. With 5’ RACE method and western blot, mRNA and protein level evidence of VDR expression is confirmed, together with differential centrifugation, the localization of VDR in leaf would be suggested to follow chlorophyll.<br/>},
  author       = {Guo, Kuo},
  isbn         = {978-91-7422-552-5},
  keyword      = {Photosynthesis,Carotenoid conversions,violaxanthin de-epoxidase (VDE),Violaxanthin de-epoxidase related protein},
  language     = {eng},
  pages        = {112},
  publisher    = {Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology},
  school       = {Lund University},
  title        = {Violaxanthin de-epoxidase and its closest relative: identification and characterization},
  year         = {2017},
}