24 research outputs found
The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders
A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders
pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?
Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst
Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase
Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage
of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor
of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel
fold with its modern descendants but displays large regions with greatly enhanced conformational
flexibility. Yet, the barrel core remains comparatively rigid and the ancestral
glycosidase activity is stable, with an optimum temperature within the experimental range for
thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures
of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase
binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding
rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the
capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular
features when sampling distant sequence space. The potential of the ancestral glycosidase as
a scaffold for custom catalysis and biosensor engineering is discussed.Uppsala Universit