
	Center for Biotechnology and Biomedicine Institute of Bioanalytical Chemistry
Structural analysis of biopolymers Prof. Dr. Norbert Sträter

Bacterial 5'-nucleotidases: Purine salvage and interference with purinergic signaling

Homologs of mammalian ectonucleotidases also exist in bacteria and other microorganisms, including many human pathogens. These enzymes have been demonstrated as virulence factors, probably by either hydrolyzing extracellular nucleotides for purine salvage or by generating extracellular adenosine in the host, which is a powerful immunosuppressant signaling molecule. NTPDases as well as CD73 homologs have been found in many microrganisms.
First structural information on 5'-nucleotidase (5NT) became available by the crystal structure of E. coli 5NT, a zinc-containing enzyme.

We characterized the structure of 5NT in open and closed conformations which differ in the relative orientation of the two domains by a rotation of up to 96° [1-4]. The domain movement can be described as a rotation of the C-terminal domain around an axis, which passes through the center of the C-terminal domain. The resulting hinge-bending domain movement is unique in that the cleft between the domains does not open up, but the residues of the domain interface slide along the interface.

Domain rotation of E. coli 5NT

Active site of E. coli 5NT with the dimetal center and the substrate binding pocket

The dimetal center of 5NT is located in the N-terminal domain (yellow) while the C-terminal domain (orange) provides the substrate binding pocket [2,6]. The conformational change observed in the crystal structures is likely to be necessary for the catalytic action of the enzyme, presumably to allow for substrate binding and product release.

Currently we study the relation between protein structure, function and the domain motion of 5NT employing especially NMR and EPR spectroscopy. We are also interested in the structure of 5'-nucleotidases from other human pathogens.

Coworkers

Thomas Knöfel
Robert Schultz-Heienbrok
Miriam Carl
Susanne Moschütz
Renato Weiße
Matthias Zebisch
Ulrike Krug
Michel Krauß
Troels E. Linnet

Collaborations

Professor Dr. Jochen Balbach, Martin-Luther-Universität Halle-Wittenberg, Biophysik
Professor Dr. Hassane Mchaourab, Vanderbilt University, Molecular Physiology & Biophysics
Professor Dr. Jens Meiler, Vanderbilt University, Computational Chemical and Structural Biology

Publications

T. Knöfel and N. Sträter (1999). X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. Nature Struct. Biol., 6, 448-453.
T. Knöfel and N. Sträter (2001). Mechanism of hydrolysis of phosphate esters by the dimetal center of 5'-nucleotidase based on crystal structures. J. Mol. Biol., 309, 239-254.
T. Knöfel and N. Sträter (2001). E. coli 5'-nucleotidase undergoes a hinge-bending domain rotation resembling a ball-and-socket motion. J. Mol. Biol., 309, 255-266
R. Schultz-Heienbrok, T. Maier, N. Sträter (2004). Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges. Protein Science, 13, 1811-1822.
R. Schultz-Heienbrok, T. Maier, N. Sträter (2005). A large Hinge Bending Domain Rotation is Required for the Catalytic Function of E. coli 5'-nucleotidase. Biochemistry, 44, 2244-2252.
U. Krug, R. Patzschke, M. Zebisch, J. Balbach, N. Sträter (2013). Contribution of the two domains of E. coli 5'-nucleotidase to substrate specificity and catalysis. FEBS letters, 587, 460-466.
U. Krug, N.S. Alexander, R.A. Stein, A. Keim, H.S. Mchaourab, N. Sträter, J. Meiler (2016). Characterization of the Domain Orientations of E. coli 5'-Nucleotidase by Fitting an Ensemble of DEER Distance Distributions. Structure, 24, 43-56.