Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.

Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-Å resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.The most frequently used cofactors in enzymatic redox reactions are the pyridine (NAD and NADP) and the flavin (FAD and FMN) nucleotides. Although NAD and NADP are soluble cofactors used by dehydrogenases, FAD and FMN usually work as prosthetic groups in flavoproteins to which they are tightly bound. Flavodoxins are small monomeric flavoproteins (15-22 kDa) that noncovalently bind a single FMN molecule, acting as a redox center (1). The flavodoxin scaffold contributes to the mechanism of electron transfer by stabilizing the FMN molecule in an environment that promotes the highly negative redox potential required for its biochemical activity. This is accomplished by sandwiching the FMN ring between two hydrophobic side chains. Flavodoxins are involved in a variety of electron transfer reactions that are essential in the metabolism of pyruvate (2), nitrogen, and pyridine nucleotides (3). These enzymes exist under three redox states: oxidized; partially reduced (semiquinone); and fully reduced (hydroquinone). Until now, flavodoxins have been identified in many prokaryotes and also in some eukaryotic algae (4 -6). In mammalian systems, flavodoxins have only been found as domains inserted in larger redox proteins such as cytochrome P450 reductase where they act as the electron transport intermediate between FAD and the P450 iron center (7). Flavodoxin-like domains are also found in mammalian nitric-oxide synthase (8) and in human erythrocyte NADPH-flavin reductase (9).The Saccharomyces cerevisiae YLR011w open reading frame codes for a protein of unknown function that has been found to be up-regulated in response to low temperature exposure (10). The sequence belongs to a COG4530 (Cluster of Orthologous Genes) predicted to generally contain flavoproteins. Hence, the YLR011wp 21-kDa protein was proposed to be composed of a single domain present in NADPH-dependent FMN reductases. We have solved the 2-Å resolution crystal structure, ...

Phosphatidylinositol (PI)1 and its phosphorylated derivatives regulate many biological processes, including cell proliferation, cell survival, differentiation, signal transduction, cytoskeleton organization, and membrane trafficking (reviewed in Ref. 1). Various chemical species can be generated by single, double, or triple phosphorylations at the inositol hydroxy groups at positions 3, 4, and 5. Their synthesis and cellular concentrations are regulated by specific lipid kinases and phosphatases. One of the major mechanisms by which PIs regulate cellular processes is by their capacity to serve as membrane signals to affect intracellular localizations of effector proteins.