Substrate and Metal Complexes of 3-Deoxy-d-arabino-heptulosonate-7-phosphate Synthase from Saccharomyces cerevisiae Provide New Insights into the Catalytic Mechanism

Abstract The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30–Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure–function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins.

Download Full-text

The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2479 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2479-2485 ◽

Cited By ~ 53

Author(s):

B S Loewy ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Regulatory Genes ◽

Wild Type ◽

Inositol 1 ◽

Cytoplasmic Enzyme ◽

Pleiotropic Phenotype ◽

Reduced Rate ◽

Phosphate Synthase

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.

Download Full-text

The two 3-deoxy- d - arabino -heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Archives of Microbiology ◽

10.1007/s002030050605 ◽

1998 ◽

Vol 169 (6) ◽

pp. 517-524 ◽

Cited By ~ 19

Author(s):

Georg Schnappauf ◽

Markus Hartmann ◽

M. Künzler ◽

G. H. Braus

Keyword(s):

Saccharomyces Cerevisiae ◽

Phosphate Synthase

Download Full-text

Catalyzed Complex Metal Hydrides

MRS Bulletin ◽

10.1557/mrs2002.227 ◽

2002 ◽

Vol 27 (9) ◽

pp. 712-716 ◽

Cited By ~ 102

Author(s):

Borislav Bogdanović ◽

Gary Sandrock

Keyword(s):

Transition Metal ◽

Metal Complexes ◽

Transition Metal Complexes ◽

Historical Perspective ◽

Catalytic Mechanism ◽

Metal Hydrides ◽

Complex Metal ◽

Sodium Alanate ◽

Storage Media ◽

Complex Hydrides

AbstractComplex hydrides are mixed ionic–covalent compounds that can serve as reversible H2 storage media only when they are catalyzed by a transition metal such as Ti. As the prime example, the phenomenology of Ti-catalyzed sodium alanate (NaAlH4) is reviewed from a historical perspective. Dehydriding yields a theoretical 5.6 wt% H2 during two-step decomposition, NaAlH4 → Na3AlH6 → NaH + Al, although 100% recovery of that H2 is not currently possible. H2 can be discharged and recharged at practical rates at 125°C. More work is needed on the alanates, in particular, as well as the identification and optimization of the catalytic mechanism and a broad extension of the concept to other than Na-based alanates. The possibility of an even further extension of the concept to other complex hydrides (e.g., the borohydrides and transition-metal complexes) is discussed.

Download Full-text

Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3320-3328.1986 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3320-3328

Author(s):

J P Hirsch ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Blot Hybridization ◽

Constitutive Expression ◽

Genomic Clone ◽

Phospholipid Synthesis ◽

Wild Type ◽

Inositol 1 ◽

Regulatory Link ◽

In Cells ◽

Phosphate Synthase

The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids. The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis. RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb. The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase. Complementation analysis showed that this transcript encoded the INO1 gene product. The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together. The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy. The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol. The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine. In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance. Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids. The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.

Download Full-text