Biosyntéza cytokininů u cyanobakterie Nostoc 7120

Bc. Helena DOBIÁŠOVÁ

Master's thesis

Cytokinin biosynthesis in cyanobacterium Nostoc 7120

Abstract:

Cytokininy jsou rostlinné hormony ovlivňující v rostlině celou řadu fyziologických procesů. První krok de novo biosyntézy cytokininů je katalyzován enzymem adenylátdimethylallyltransferasou (IPT), který je přítomen jak v rostlinách, tak i v některých bakteriích. Gen (NoIPT1) kódující IPT byl nalezen v genomu cyanobakterie Nostoc PCC 7120. Zmíněný gen byl vyizolován a klonován do vektoru pTYB12, ale purifikace exprimovaného proteinu byla neúspěšná. Proto byl tento gen z vektoru pTYB12 klonován do vektoru pET-28b(+) a opět exprimován v Escherichia coli. Purifikačním procesem nebylo dosaženo homogenity proteinu NoIPT1. Po zjištění, že se jedná o aktivní enzym, byla aktivita enzymu stanovována pomocí metody HPLC/UV. Pro enzym NoIPT1 bylo určeno pH optimum (pH 7,5). Dále bylo prokázáno, že tento enzym zajišťuje přenos isopentenylové skupiny z dimethylallyldifosfátu (DMAPP) na adenosinmonofosfát (AMP). Pro DMAPP a AMP byly stanoveny Michaelisovy konstanty. Afinita enzymu nebyla prokázána pro sloučeniny isopentenyldifosfát (iPP) a 1-hydroxy-2-methyl-2-(E)-butenyl-4-difosfát (HMBDP). Zároveň ale nebyly nalezeny jednoznačné důkazy, že enzym není schopen prenylovat nukleotidy adenosindifosfát (ADP) a adenosintrifosfát (ATP). V následujících experimentech budou hledány jiné způsoby purifikace enzymu, aby bylo dosaženo jeho vysoké čistoty. Enzym také bude podrobněji charakterizován.

Abstract:

Cytokinins are the plant hormones affecting a series of physiological processes in plants. The first step of the de novo biosynthesis of cytokinins is catalyzed by an adenylate dimethylallyltransferase (IPT), which is present both in the plants and some bacteria. The gene (NoIPT1) encoding putative IPT was identified in the genome of cyanobacterium Nostoc PCC 7120. The gene was isolated and cloned into the vector pTYB12, but the purification of the expressed protein was not successful. The gene was therefore cloned from the vector pTYB12 into the vector pET-28b(+) and was expressed in Escherichia coli. Although the protein purification process was extensively optimized, obtained NoIPT1 protein was not homogenous. After finding that the enzyme was active the enzyme activity was determined using HPLC/UV method. The pH optimum of NoIPT1 was determined to be 7.5. It was also shown that the enzyme transfers isopentenyl group from dimethylallyl diphosphate (DMAPP) to adenosine monophosphate (AMP). The Michaelis constants for DMAPP and AMP were determined. The affinity of the enzyme was not confirmed for the compounds isopentenyl diphosphate (iPP) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP). At the same time, the conclusive evidence that the enzyme is not able to bind adenosine diphosphate (ADP) and adenosine triphosphate (ATP) nucleotides was not found. In the following experiments, other purification methods will be examined to achieve protein homogenity and the enzyme will be characterized in more detail.

Keywords

Nostoc PCC 7120, cytokinin, NoIPT1, DMAPP, HMBDP

Language used: Czech

Date on which the thesis was submitted / produced: 30. 4. 2013

Accessible from:: 30. 4. 2013

Thesis defence

• Date of defence: 27. 5. 2013
• Supervisor: Doc. RNDr. Jitka Frébortová, Ph.D.

PALACKÝ UNIVERSITY IN OLOMOUC

Faculty of Science

Master programme / field:
Biochemistry / Biochemistry