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1.  A conserved asparagine plays a structural role in ubiquitin-conjugating enzymes 
Nature chemical biology  2013;9(3):154-156.
It is widely accepted that ubiquitin conjugating enzymes (E2) contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13/Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.
PMCID: PMC3578109  PMID: 23292652
2.  A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins1 
Analytical biochemistry  2011;418(1):102-110.
Ubiquitination is a widely-studied regulatory modification involved in protein degradation, DNA damage repair and the immune response. Conjugation of ubiquitin to a substrate lysine occurs in an enzymatic cascade involving an E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, or E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
PMCID: PMC3178097  PMID: 21771579
Ubiquitin Conjugation; Ubiquitin-like proteins; Ubc13-Mms2; ubiquitin-activating enzyme
3.  Catalytic mechanism of a MYST family histone acetyltransferase † 
Biochemistry  2007;46(3):623-629.
Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferases (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-ε-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first, and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 × 10−4 s−1, or ∼2500-fold slower than histone acetylation, kcat =1.6 s−1) and of CoA formation (0.0021 s−1) were inconsistent with a kinetically competent acetylated-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from wild-type. Similarly, a pH rate (kcat) analysis of wild-type and C304A revealed an ionization (pKa = 7.6−7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat ∼200-fold at pH 7.5; however at higher pH, the E338Q displayed nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-ε-lysine by deprotonation. Together the results suggest that MYST family HATs utilize a direct-attack mechanism within a ternary complex of Esa1•acetyl-CoA•histone.
PMCID: PMC2752042  PMID: 17223684
4.  Catalysis and Substrate Selection by Histone/Protein Lysine Acetyltransferases 
Reversible protein acetylation is controlled by the opposing actions of protein lysine acetyltransferases and deacetylations. Recent developments on the structure and biochemical mechanisms of histone acetyltransfers (HATs) have provided new insight into catalysis and substrate selection. Diverse families of HATs appear to perform a conserved mechanism of acetyl-transfer, where the lysine-containing substrate directly attacks enzyme-bound acetyl-CoA. The ability of HATs to form distinct multi-subunit complexes provide a means to regulate HAT activity by altering substrate specificity, targeting to specific loci, enhancing acetyltransferase activity, restricting access of non-target proteins, and coordinating the multiple enzyme activities of the complex. In the case of newly discovered Rtt109 HAT, association with distinct histone chaperones directs substrate selection between N-terminal lysines (H3K9, H3K23) and those (H3K56) within the histone fold domain. Moreover, the ability of some HATs to utilize longer chain acyl-CoA (i.e. propionyl-CoA) as alternative substrates suggests a potential direct link between the metabolic state of the cell and transcriptional regulation.
PMCID: PMC2723715  PMID: 19056256
5.  Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75 
Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by ∼100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the In vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.
PMCID: PMC2678805  PMID: 19172748
6.  Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family †,‡ 
Biochemistry  2006;45(48):14325-14336.
We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (Rfree = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15N and 13C+15N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8–93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540–CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys76 and His41 residues of this family.
PMCID: PMC2533278  PMID: 17128971
Molecular cell  2007;25(5):703-712.
Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone-binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109-Asf1 complex but not the Rtt109-Vps75 complex being critical for resistance to genotoxic agents.
PMCID: PMC1853276  PMID: 17320445
8.  Nucleosome Recognition by the Piccolo NuA4 Histone Acetyltransferase Complex† 
Biochemistry  2007;46(8):2091-2099.
The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21–52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be “tethered”, thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation.
PMCID: PMC1994252  PMID: 17274630

Results 1-8 (8)