MTD 243-670 Instrukcja Użytkownika Pobierz pdf (Strona 7)

MtrA from M. jannaschii without its transmembrane helix could be produced in

E. coli as a soluble protein. The holoprotein could be purified to homogeneity but

crystallization failed presumably due to its exceptionally high solubility. MtrA from

M. kandleri was produced in E. coli as a StrepII fusion protein without

transmembrane helix only in marginal amounts.

The production of subunit MtrH in E. coli as a soluble protein was not possible

regardless of the variants tested in this thesis. Attempts to refold and purify to

homogeneity the M. marburgensis protein expressed in inclusion bodies were without

success. Co-expression of MtrA and MtrH with the objective of improving folding and

solubility also led to the production of inclusion bodies which could not be refolded

and purified together.

The second enzyme analyzed in this thesis, F

420

-dependent N

-methylene-

MPT dehydrogenase (Mtd), catalyzes the reversible stereospecific hydride transfer

between reduced F

420

) and methenyl-H

MPT

, the latter being thereby

reduced to methylene-H

MPT. The ternary complex involved in this reaction consists

of the protein part, substrate (methylene-H

MPT) and co-substrate (F

420

) and was

structurally characterized in this thesis. The purified recombinant enzyme from

M. kandleri was co-crystallized with several H

MPT and F

420

derivatives, the structure

of the ternary complex was determined by X-ray crystallography and the binding of

MPT and F

420

was analyzed. In the structure solved in the thesis methenyl-H

MPT

and F

420

are bound in a catalytically active conformation, but a resolution of 1.8 Å

precludes a discrimination between either methylene-H

MPT and F

420

or methenyl-

MPT

and F

420

. Compared to the structure of M. kandleri Mtd (KMtd) without

substrate and co-substrate bound, only marginal variations of the protein

conformation were visible. Thus KMtd can be considered as a surprising and extreme

example of an enzyme with an exceptionally rigid, preformed binding pocket.

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