TY  - JOUR
A1  - Haenzelmann, Petra
A1  - Dahl, Jan U.
A1  - Kuper, Jochen
A1  - Urban, Alexander
A1  - Mueller-Theissen, Ursula
A1  - Leimkühler, Silke
A1  - Schindelin, Hermann
T1  - Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains
N2  - Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well- characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other gamma-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3- mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.
Y1  - 2009
UR  - http://www.proteinscience.org/
U6  - https://doi.org/10.1002/pro.260
SN  - 0961-8368
ER  - 
TY  - JOUR
A1  - Dietzel, Uwe
A1  - Kuper, Jochen
A1  - Doebbler, Jennifer A.
A1  - Schulte, Antje
A1  - Truglio, James J.
A1  - Leimkühler, Silke
A1  - Kisker, Caroline
T1  - Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase
N2  - Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6- aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.
Y1  - 2009
UR  - http://www.jbc.org/
U6  - https://doi.org/10.1074/jbc.M808114200
SN  - 0021-9258
ER  -