Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex

Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.     

Structure of an acetyl-CoA binding protein from Staphylococcus aureus representing a novel subfamily of GCN5-related N-acetyltransferase-like proteins

We have determined the solution NMR structure of SACOL2532, a putative GCN5-like N-acetyltransferase (GNAT) from Staphylococcus aureus. SACOL2532 was shown to bind both CoA and acetyl-CoA, and structures with and without bound CoA were determined. Based on analysis of the structure and sequence, a subfamily of small GCN5-related N-acetyltransferase (GNAT)-like proteins can be defined. Proteins from this subfamily, which is largely congruent with COG2388, are characterized by a cysteine residue in the acetyl-CoA binding site near the acetyl group, by their small size in relation to other GNATs, by a lack of obvious substrate binding site, and by a distinct conformation of bound CoA in relation to other GNATs. Subfamily members are found in many bacterial and eukaryotic genomes, and in some archaeal genomes. Whereas other GNATs transfer the acetyl group of acetyl-CoA directly to an aliphatic amine, the presence of the conserved cysteine residue suggests that proteins in the COG2388 GNAT-subfamily transfer an acetyl group from acetyl-CoA to one or more presently unidentified aliphatic amines via an acetyl (cysteine) enzyme intermediate. The apparent absence of a substrate-binding region suggests that the substrate is a macromolecule, such as another protein, or that a second protein subunit providing a substrate-binding region must combine with SACOL2532 to make a fully functional N-acetyl transferase.     

Computational Analysis of N-acetyl transferase in Tribolium castaneum

N-acetyl transferase (NAT) is responsible to catalyze the transfer of acetyl groups to arylamines from acetyl-CoA. Aralkylamine Nacetyl transferase (AANAT), which belongs to GCN5-related N-acetyl transferase member, is a globular 23-kDa cytosolic protein that forms a reversible regulatory complex with 14-3-3 proteins, AANAT regulates the daily cycle of melatonin biosynthesis in mammals, making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. There is no evidence available regarding α and β subunits, active site and their ASA value in Dopamine N-acetyl transferase. Therefore, we describe the development of Dopamine N-acetyl transferase model in Tribolium castaneum. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides insight to the predicted active site and ASA values of Dopamine N-acetyl transferase model in Tribolium castaneum.     

Cloning and structural analysis of the murine GCN5L1 gene

We recently cloned the murine 11-cis retinol dehydrogenase gene. A second gene, the murine GCN5L1 gene, was found to be situated upstream of the murine 11-cis retinol dehydrogenase gene. We have isolated and sequenced the complete coding sequence of the murine GCN5L1 gene. The distance between the 3′-end of the murine GCN5L1 gene and the 5′-end of the 11-cis retinol dehydrogenase gene is only 776 nt. The murine GCN5L1 gene consists of four exons encompassing approximately 3.5 kb of genomic DNA. Intron/exon splice sites conform to the GT/AG rule. The open reading frame consists of 375 nucleotides encoding a 14 kDa protein. The murine GCN5L1, like the human GCN5L1 protein, displays weak homology (27%) to yeast GCN5. The distance between the murine, human and bovine GCN5L1 and 11-cis retinol dehydrogenase genes appeared to be conserved.     

Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms

General control nonderepressible kinase 2 (GCN2) is a promising target for cancer therapy. However, the role of GCN2 in cancer cell survival or death is elusive; further, small molecules targeting GCN2 signaling are not available. By using a GCN2 level-based drug screening assay, we found that GCN2 protein level critically determined the sensitivity of the cancer cells toward Na⁺,K⁺-ATPase ligand–induced apoptosis both in vitro and in vivo, and this effect was largely dependent on C/EBP homologous protein (CHOP) induction. Further analysis revealed that GCN2 is a short-lived protein. In A549 lung carcinoma cells, cellular β-arrestin1/2 associated with GCN2 and maintained the GCN2 protein level at a low level by recruiting the E3 ligase NEDD4L and facilitating consequent proteasomal degradation. However, Na⁺,K⁺-ATPase ligand treatment triggered the phosphorylation of GCN2 at threonine 899, which increased the GCN2 protein level by disrupting the formation of GCN2–β-arrestin–NEDD4L ternary complex. The enhanced GCN2 level, in turn, aggravated Na⁺,K⁺-ATPase ligand–induced cancer cell apoptosis. Our findings reveal that GCN2 can exert its proapoptotic function in cancer cell death by posttranslational mechanisms. Moreover, Na⁺,K⁺-ATPase ligands emerge as the first identified small-molecule drugs that can trigger cancer cell death by modulating GCN2 signaling.     

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.     

Investigation of the acetylation mechanism by GCN5 histone acetyltransferase

The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes.     

On the presence of carnitine acetyl transferase in human platelets

Summary The presence of carnitine acetyl transferase (E.C.2.3.1.7) activity has been found for the first time in human platelets. The enzymic activity was measured by a radiometric method based on the separation of labelled acetylcarnitine and carnitine on a cation exchange column. Carnitine acetyl transferase activity closely paralleled the activity distribution of the mitochondrial marker carnitine palmitoyl-transferase. Contrary to the marker enzyme, human platelet carnitine acetyl-transferase is rather thermosensitive: 60% of its activity is lost after 10 min when kept at 37°C.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.