Magnesium Chelatase - a Key Enzyme in Chlorophyll Biosynthesis

Eva Axelsson

Magnesium Chelatase - a Key Enzyme in Chlorophyll Biosynthesis

Eva Axelsson

Biokemi och Strukturbiologi

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

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Popular Abstract in Swedish
Klorofyllsyntes är en viktig process i naturen. De gröna pigmenten utgör en viktig del i fotosyntesen där de absorberar och överför solenergi till fotosyntetiska reaktionscentra. Magnesium kelatas katalyserar insättandet av en magnesium (II) jon i protoporfyrin IX, vilket är det första specifika steget i klorofyllsyntesen. Magnesium kelatas består av tre proteiner, som kallas för I-, D- och H-subenheterna.

Den huvudsakliga målsättningen med den här avhandlingen har varit att karaktärisera det 70 kDa stora D-proteinet. Analys av dess aminosyra sekvens visar att proteinet innehåller en N-terminal atypisk AAA-domän (ATPases associated with various cellular activities) och en C-terminal integrin I domän. Den tre-dimensionella strukturen av I-subenheten hos Rhodobacter capsulatus har tidigare bestämts med röntgen kristallografi och elektron mikroskopering. Dessa studier har visat att proteinet bildar en hexamerisk ring struktur. I den här avhandlingen presenteras för första gången en rekonstruktion av den tre-dimensionella strukturen av ett ID-komplex vid 23 Å upplösning. Strukturen visar att proteinerna bildar ett stort komplex bestående av två hexameriska ringar. Vidare presenteras en grundläggande karaktärisering av fem recessiva korn mutanter, vilka alla är defekta i magnesium kelatas D-subenheten. Bristen av klorofyll i dessa mutanter visade sig vara orsakade av ett instabilt D-protein. D-subenheten hos R. capsulatus studerades även in vitro där det observerades att inaktiva mutanter hade en dominant effekt på magnesium kelatas aktiviteten, vilket indikerar att D-subenheterna arbetar kooperativt. Den dominanta effekten visade sig att vara beroende av hur proverna, innehållande alla magnesium kelatas proteinerna, hade preparerats före analys. Resultaten presenterade i denna avhandling föreslår att en möjlig funktion av D-subenheten är att utgöra en funktionell stabiliserande hexamerisk plattform för den mer instabila och ATP hydrolyserande I-subenheten.

En majoritet av proteinerna som är involverade i det fotosyntetiska maskineriet är kodade av gener från det nukleära genomet. Klorofyll intermediärer har tidigare visats fungera som signalmolekyler mellan kloroplast och cellkärnan. I denna avhandling presenteras även en studie angående signalering mellan kloroplast och cellkärna hos korn mutanter defekta i magnesium kelatas och magnesium protoporfyrin IX monometylester cyklas med hjälp av microarray teknologi.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution	Biokemi och Strukturbiologi
Handledare	Hansson, Mats, handledare
Tilldelningsdatum	2006 mars 10
Förlag	Eva Axelsson
ISBN (tryckt)	91-7422-107-8
Status	Published - 2006

Bibliografisk information

Defence details

Date: 2006-03-10
Time: 10:30
Place: Centre for Chemistry and Chemical Engineering Getingevägen 60, 211 00 Lund, Sweden

External reviewer(s)

Name: Smith, Alison
Title: Dr.
Affiliation: Department of Plant Sciences, University of Cambridge, U.K.

---

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title = "Magnesium Chelatase - a Key Enzyme in Chlorophyll Biosynthesis",

abstract = "The biosynthesis of chlorophylls is an important process in nature. The green pigments play a fundamental role in absorbing and transferring solar energy to the photosynthetic apparatus. This thesis elucidates the role and function of the enzyme magnesium chelatase, which catalyses the insertion of magnesium (II) into protoporphyrin IX and is the first committed enzyme of the chlorophyll biosynthetic pathway. Magnesium chelatase consists of three proteins, referred to as the I-, D- and H-subunits. In this thesis, the main focus has been to characterize the intermediate sized 70 kDa D-subunit. Previously, the crystal structure of R. capsulatus I-subunit was determined and electron microscopy analysis revealed that the protein formed a hexameric ring structure. The protein was found to belong to the AAA (ATPases associated with various cellular activities) family of proteins. Analysis of the amino acid sequence of the D-subunit suggests that the protein contain an atypical AAA-domain in the N-terminus and an integrin I domain in the C-terminus, which is linked by a proline rich and negatively charged region. In paper II, we present the structure of the R. capsulatus ID-complex of magnesium chelatase at 23 {\AA} resolution by negative staining electron microscopy. The structure demonstrates that the proteins form a large complex of two hexameric rings. In paper I, we have characterized five recessive barley mutants defective in the D-subunit of magnesium chelatase and it was concluded that an unstable D-protein causes chlorophyll deficiency in vivo. The D-protein was further investigated in vitro by using heterologously expressed Rhodobacter capsulatus BchD. In vitro, inactive mutants exerted a dominant effect on magnesium chelatase activity, indicating that the D-proteins are cooperative and play an active role during catalysis. However, in paper III it was demonstrated that the dominant effect was dependant on the preparation of protein samples prior analysis. These observations lead to further investigations and discussions concerning the assembly and stability of the D-protein itself and in complex with the I-protein. We hypothesize that in the ID-complex, the D-protein form a stable hexameric platform for the unstable and ATPase active I-protein. A majority of the proteins involved in the photosynthetic apparatus are encoded by genes of the nuclear genome. Intermediates of the chlorophyll biosynthetic pathway have been found to function as signal molecules in chloroplast-to-nucleus signal transduction. In paper IV we investigated the signal transduction of barley mutants defective in magnesium chelatase and magnesium protoporphyrin IX monomethylester cyclase by using microarray technology.",

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N2 - The biosynthesis of chlorophylls is an important process in nature. The green pigments play a fundamental role in absorbing and transferring solar energy to the photosynthetic apparatus. This thesis elucidates the role and function of the enzyme magnesium chelatase, which catalyses the insertion of magnesium (II) into protoporphyrin IX and is the first committed enzyme of the chlorophyll biosynthetic pathway. Magnesium chelatase consists of three proteins, referred to as the I-, D- and H-subunits. In this thesis, the main focus has been to characterize the intermediate sized 70 kDa D-subunit. Previously, the crystal structure of R. capsulatus I-subunit was determined and electron microscopy analysis revealed that the protein formed a hexameric ring structure. The protein was found to belong to the AAA (ATPases associated with various cellular activities) family of proteins. Analysis of the amino acid sequence of the D-subunit suggests that the protein contain an atypical AAA-domain in the N-terminus and an integrin I domain in the C-terminus, which is linked by a proline rich and negatively charged region. In paper II, we present the structure of the R. capsulatus ID-complex of magnesium chelatase at 23 Å resolution by negative staining electron microscopy. The structure demonstrates that the proteins form a large complex of two hexameric rings. In paper I, we have characterized five recessive barley mutants defective in the D-subunit of magnesium chelatase and it was concluded that an unstable D-protein causes chlorophyll deficiency in vivo. The D-protein was further investigated in vitro by using heterologously expressed Rhodobacter capsulatus BchD. In vitro, inactive mutants exerted a dominant effect on magnesium chelatase activity, indicating that the D-proteins are cooperative and play an active role during catalysis. However, in paper III it was demonstrated that the dominant effect was dependant on the preparation of protein samples prior analysis. These observations lead to further investigations and discussions concerning the assembly and stability of the D-protein itself and in complex with the I-protein. We hypothesize that in the ID-complex, the D-protein form a stable hexameric platform for the unstable and ATPase active I-protein. A majority of the proteins involved in the photosynthetic apparatus are encoded by genes of the nuclear genome. Intermediates of the chlorophyll biosynthetic pathway have been found to function as signal molecules in chloroplast-to-nucleus signal transduction. In paper IV we investigated the signal transduction of barley mutants defective in magnesium chelatase and magnesium protoporphyrin IX monomethylester cyclase by using microarray technology.

AB - The biosynthesis of chlorophylls is an important process in nature. The green pigments play a fundamental role in absorbing and transferring solar energy to the photosynthetic apparatus. This thesis elucidates the role and function of the enzyme magnesium chelatase, which catalyses the insertion of magnesium (II) into protoporphyrin IX and is the first committed enzyme of the chlorophyll biosynthetic pathway. Magnesium chelatase consists of three proteins, referred to as the I-, D- and H-subunits. In this thesis, the main focus has been to characterize the intermediate sized 70 kDa D-subunit. Previously, the crystal structure of R. capsulatus I-subunit was determined and electron microscopy analysis revealed that the protein formed a hexameric ring structure. The protein was found to belong to the AAA (ATPases associated with various cellular activities) family of proteins. Analysis of the amino acid sequence of the D-subunit suggests that the protein contain an atypical AAA-domain in the N-terminus and an integrin I domain in the C-terminus, which is linked by a proline rich and negatively charged region. In paper II, we present the structure of the R. capsulatus ID-complex of magnesium chelatase at 23 Å resolution by negative staining electron microscopy. The structure demonstrates that the proteins form a large complex of two hexameric rings. In paper I, we have characterized five recessive barley mutants defective in the D-subunit of magnesium chelatase and it was concluded that an unstable D-protein causes chlorophyll deficiency in vivo. The D-protein was further investigated in vitro by using heterologously expressed Rhodobacter capsulatus BchD. In vitro, inactive mutants exerted a dominant effect on magnesium chelatase activity, indicating that the D-proteins are cooperative and play an active role during catalysis. However, in paper III it was demonstrated that the dominant effect was dependant on the preparation of protein samples prior analysis. These observations lead to further investigations and discussions concerning the assembly and stability of the D-protein itself and in complex with the I-protein. We hypothesize that in the ID-complex, the D-protein form a stable hexameric platform for the unstable and ATPase active I-protein. A majority of the proteins involved in the photosynthetic apparatus are encoded by genes of the nuclear genome. Intermediates of the chlorophyll biosynthetic pathway have been found to function as signal molecules in chloroplast-to-nucleus signal transduction. In paper IV we investigated the signal transduction of barley mutants defective in magnesium chelatase and magnesium protoporphyrin IX monomethylester cyclase by using microarray technology.

KW - Kemi

KW - Chemistry

KW - AAA-proteins

KW - Chlorophyll biosynthesis

KW - Magnesium chelatase

M3 - Doctoral Thesis (compilation)

SN - 91-7422-107-8

PB - Eva Axelsson

ER -

Magnesium Chelatase - a Key Enzyme in Chlorophyll Biosynthesis

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