Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling.

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites.

The origin of 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) in the lipopolysaccharide of Shewanella oneidensis.

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis.

Structure of the metal-dependent deacetylase LpxC from Yersinia enterocolitica complexed with the potent inhibitor CHIR-090 .

The first committed step of lipid A biosynthesis is catalyzed by UDP-(3-O-((R)-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase, a metal-dependent deacetylase also known as LpxC. Because lipid A is essential for bacterial viability, the inhibition of LpxC is an appealing therapeutic strategy for the treatment of Gram-negative bacterial infections. Here we report the 1.

Active site metal ion in UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) switches between Fe(II) and Zn(II) depending on cellular conditions.

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6-8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M.

Structures of metal-substituted human histone deacetylase 8 provide mechanistic inferences on biological function .

The metal-dependent histone deacetylases (HDACs) adopt an alpha/beta protein fold first identified in rat liver arginase. Despite insignificant overall amino acid sequence identity, these enzymes share a strictly conserved metal binding site with divergent metal specificity and stoichiometry. HDAC8, originally thought to be a Zn(2+)-metallohydrolase, exhibits increased activity with Co(2+) and Fe(2+) cofactors based on k(cat)/K(M) (Gantt, S.

Activation of Escherichia coli UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase by Fe2+ yields a more efficient enzyme with altered ligand affinity.

The metal-dependent deacetylase UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate. Consequently, LpxC is a target for the development of antibiotics, nearly all of which coordinate the active site metal ion. Here we examine the ability of Fe(2+) to serve as a cofactor for wild-type Escherichia coli LpxC and a mutant enzyme (EcC63A), in which one of the ligands for the inhibitory metal binding site has been removed.

Mechanism of dihydrouridine synthase 2 from yeast and the importance of modifications for efficient tRNA reduction.

Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this DUS were very low.

Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors.

Metal-dependent histone deacetylases (HDACs) require Zn(2+) or Fe(2+) to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. Isozyme HDAC8 is perhaps the archetypical member of the class I HDAC family and serves as a paradigm for studying structure-function relationships. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide (APHA) in a new crystal form.

Catalytic activity and inhibition of human histone deacetylase 8 is dependent on the identity of the active site metal ion.

Histone deacetylases play a key role in regulating transcription and other cellular processes by catalyzing the hydrolysis of epsilon-acetyl-lysine residues. For this reason, inhibitors of histone deacetylases are potential targets for the treatment of cancer. A subset of these enzymes has previously been shown to require divalent metal ions for catalysis.

Direct observation of the participation of flavin in product formation by thyX-encoded thymidylate synthase.

The synthesis of thymine for DNA is catalyzed by the enzyme thymidylate synthase (TS). A family of flavin-dependent TSs encoded by the thyX gene has been discovered recently. These newly discovered TSs require a reducing substrate in addition to 2'-deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (CH2THF), suggesting that the enzyme-bound flavin is a redox intermediary in catalysis.