Universität zu Köln

Havarushka, Nastassia, Fischer-Schrader, Katrin ORCID: 0000-0003-4532-8918, Lamkemeyer, Tobias and Schwarz, Guenter ORCID: 0000-0002-2118-9338 (2014). Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from Pyrococcus furiosus. PLoS One, 9 (1). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1932-6203

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Abstract

Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 angstrom resolution and identified key residues of alpha 3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 degrees C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 degrees C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Havarushka, Nastassia UNSPECIFIED UNSPECIFIED UNSPECIFIED Fischer-Schrader, Katrin UNSPECIFIED orcid.org/0000-0003-4532-8918 UNSPECIFIED Lamkemeyer, Tobias UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwarz, Guenter UNSPECIFIED orcid.org/0000-0002-2118-9338 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-448380
DOI:	10.1371/journal.pone.0086030
Journal or Publication Title:	PLoS One
Volume:	9
Number:	1
Date:	2014
Publisher:	PUBLIC LIBRARY SCIENCE
Place of Publication:	SAN FRANCISCO
ISSN:	1932-6203
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; CONFORMATIONAL FLEXIBILITY; TRIOSEPHOSPHATE ISOMERASE; GLUTAMATE-DEHYDROGENASE; MOLYBDENUM INSERTION; ENZYME STABILITY; KEY ROLE; THERMOSTABILITY; MOLYBDOPTERIN Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/44838