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SUMMARY

The aim of this thesis was a structural characterization of the binding of

tetrahydromethanopterin derivatives to enzymes of the energy conserving CO

reducing methanogenic pathway. In this pathway the stepwise reduction of CO

proceeds via binding to the tetrahydrofolate analog tetrahydromethanopterin

MPT) which is found i. a. in methanogenic archaea. Due to the adaptation of the

thermophilic and hyperthermophilic source organisms (Methanothermobacter

marburgensis, Methanocaldococcus jannaschii and Methanopyrus kandleri) to their

extreme habitats by genomic, structural and enzymatic features, they are of special

interest for structural biology.

The first enzyme investigated in this thesis is the eight subunits containing

membrane bound complex of N

-methyl-H

MPT:coenzyme M methyltransferase

(MtrA-H). Firstly, it catalyzes the methyl group transfer from H

MPT to the Co(I) of the

prosthetic group (5’-hydroxybenzimidazolylcobamide; vitamin B

12a

). In a second step,

it transfers the methyl group to coenzyme M, which is coupled to an energy

conserving vectorial sodium ion transport across the membrane.

The purification protocol for Mtr complex from M. marburgensis (670 kDa)

previously established under anaerobic conditions was enhanced, simplified for

isolation and purification under aerobic conditions and optimized for electron

microscopic single particle reconstruction. Besides the preparation of the complete

complex MtrA-H, the preparation of enzyme complex MtrA-G without the most

hydrophilic subunit MtrH was chosen as a second approach. The purification method

developed for this purpose improved the control over dissociation of MtrH from

complex MtrA-G and enhanced the homogeneity of the sample significantly. Thus,

the prerequisites for crystallization and subsequent X-ray studies were created as

well as for electron microscopic single particle reconstruction, which was confirmed

by experiments with MtrA-G (without MtrH) promising far better results.

Concurrently to the studies on the complete Mtr complex, cobamide containing

subunit MtrA and H

MPT binding subunit MtrH should be purified to homogeneity in

quantities sufficient for crystallization and X-ray analysis. Therefore, MtrA and MtrH

from source organisms mentioned above were cloned for heterologous expression in

E. coli, expression conditions were optimized and purification protocols were

established. The purified proteins were used for extensive crystallization

experiments.

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MTD 243-670 Manual do Utilizador Página 6