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Structural and functional studies on the CysC domain of ATP sulfurylase in Mycobacterium tuberculosis
KTH, School of Biotechnology (BIO).
2011 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Strukturella och funktionella studier av CysC domänen i ATP sulfurylase i Mycobacterium tuberculosis (Swedish)
Abstract [en]

Estimates suggest that 2 billion people are infected with latent tuberculosis, bearing a huge reservoir for acute infections [1], since the inability of the host immune system to kill Mycobacterium tuberculosis (Mtb) effectively allows pathogenic Mtb cells to survive inside macrophages [2]. The importance of studying the metabolism of Mtb during latent infections results from observed reactivation, and development of acute tuberculosis in approximately 10 % of all latent tuberculosis cases [3]. Stress stimuli, mimicking the conditions inside a macrophage during a latent infection  induce amongst others the expression of genes encoding [4] for the tri-functional ATP sulfurylase enzyme complex. The APS kinase domain (CysC), which is one functional constitutent of ATP sulfurylase, catalyzes the phosphorylation of adenosine-5’-phosphosulfate (APS) under consumption of adenosine 5 0-triphosphate ATP) to formadenosine 3-phosphate 5-phosphosulfate (PAPS), which is the sole substrate for sulfotransferases to produce sulfolipid 1, a major contributor to Mtb’s virulence [5]. In this project structural and functional studies on CysC of Mtb have been performed. CysC was heterologously expressed in E. coli BL21(DE3), and purified to homogeneity. Kinase activity of CysC has been assessed after establishing a method based on anion exchange thin layer chromatography (TLC) to separate the substrates and products of the CysC catalyzed reaction that afterwards were detected by UV light. This approach to activity assessment showed that CysC is active, although the enzyme is expressed separately from its native fusion partner, the GTPase CysN and reveals that fusion of CysC and CysN is no prerequisite for kinase activity of CysC in Mtb.

Crystals of CysC in complex with its native substrate APS, and the non-hydrolysable ATP analog, AMP-PNP, have been obtained by co-crystallization. An X-ray dataset was collected to a resolution of 1.8 Å and the obtained electron density maps indicate that the two ligands are bound to the enzyme active site. Furthermore the data suggests that the highly conserved DGDN-loop undergoes a conformational change.

Abstract [sv]

Uppskattningar av WHO tyder på att 2 miljarder människor är infekterade med latent tuberkulos, vilket är en enorm reservoar för akuta infektioner [1]. Begränsningarna i värdens immunsystems förmåga att effektivt döda Mycobacterium tuberculosis(Mtb) leder till att patogenen kan överleva inuti makrofager [2], vilket kan resultera i reaktivering och utveckling av akut tuberkulos hos ungefär 10% av alla latenta tuberkulos fall [3]. In vivo odeller som imiterar förhållandena inuti en makrofag under en latent infektion medför bland annat uttrycket av gener som kodar [4] för det tri-funktionella ATP sulfurylase enzym komplexet. Detta protein komplex består av tre olika enzym, CysD, en adenylyl-transferas, GTPaset CysN och APS kinaset CysC. I många mikroorganismer kodas dessa proteiner av tre gener, emedan CysN och CysC i Mb består av ett fusionsprotein innehållande båda domänerna. CysC katalyserar fosforyleringen av adenosin-5'- phosphosulfat (APS) under konsumtion av adenosin 5'-trifosfat (ATP) för att bilda adenosin 3'- fosfat 5 ' -phosphosulfat (PAPS). Produkten PAPS är substratet för sulfotransferaser som leder till bildning av sulfolipid 1, en beståndsdel i bakteriens cellvägg och en viktig virulensfaktor [5]. I detta projekt har strukturella och funktionella studier av CysC från Mtb utförts. Rekombinant CysC har uttryckts i E. coli BL21 (DE3) och framställts i ren form. Kinas aktivitet av CysC har kunnat påvisas med hjälp av tunnskiktskromatografi (TLC) vilket visar att den rekombinanta CysC domänen är katalytiskt aktiv som kinas också i frånvaro av CysN domänen och CysD.

Kristaller av CysC i komplex med dess substrat APS och den icke-hydrolyserbara ATP analogen AMP-PNP har framställts genom ko-kristallisering. Röntgendata samlades till en upplösning av 1.8 Å och de erhållna elektrontäthetskartorna visar att båda ligander binds till enzymets aktiva yta. Vidare tyder de erhållna data på att den konserverade loopen -DGDN- genomgår en konformationsändring som en del i enzymets mekanism.

Place, publisher, year, edition, pages
2011.
Keyword [en]
Mycobacterium tuberculosis, sulfur metabolism, protein structure, structural biology, enzymology
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-49074OAI: oai:DiVA.org:kth-49074DiVA: diva2:459241
Subject / course
Biotechnology
Educational program
Degree of Master
Uppsok
Technology
Supervisors
Examiners
Available from: 2011-11-25 Created: 2011-11-25 Last updated: 2011-11-25Bibliographically approved

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