Studies of the in vitro Nalpha-acetyltransferase activities of E. coli RimL protein

Although the Escherichia coli N(alpha)-acetyltransferase RimL catalyzing the N-terminal acetylation of L12 have been identified through mutant analysis, little is known about its enzymatic activity and auxiliary subunit requirement. This study was to investigate the enzymatic activities of RimL and its substrate specificity. RimL, its substrate L12, and two mutant substrates L12S1A and L12I2D were overexpressed and purified from E. coli. In vitro experimental results revealed that RimL itself can convert L12 to L7 by acetylation of the N-terminal serine residue. The K(m) value for L12 was 0.55 microM and the V(max) was 25.71 min(-1) as determined by a spectrophotometrical method. We also found that RimL acetylated the L12S1A mutant with an N-terminal alanine residue instead of the native serine residue, suggesting RimL can acetylate other N-terminal residues. Furthermore, when the second N-terminal residue isoleucine was replaced by aspartic acid, the mutant L12I2D was also acetylated by RimL but under a much lower rate.     

Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation

The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets. We report the x-ray crystal structure of Naa50p bound to a native substrate peptide fragment and CoA. We found that the peptide backbone of the substrate is anchored to the protein through a series of backbone hydrogen bonds with the first methionine residue specified through multiple van der Waals contacts, together creating an α-amino methionine-specific pocket. We also employed structure-based mutagenesis; the results support the importance of the α-amino methionine-specific pocket of Naa50p and are consistent with the proposal that conserved histidine and tyrosine residues play important catalytic roles. Superposition of the ternary Naa50p complex with the peptide-bound Gcn5 histone acetyltransferase revealed that the two enzymes share a Gcn5-related N-acetyltransferase fold but differ in their respective substrate-binding grooves such that Naa50p can accommodate only an α-amino substrate and not a side chain lysine substrate that is acetylated by lysine acetyltransferase enzymes such as Gcn5. The structure of the ternary Naa50p complex also provides the first molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications.     

Yeast MAK3 N-acetyltransferase recognizes the N-terminal four amino acids of the major coat protein (gag) of the L-A double-stranded RNA virus.

The MAK3 gene of Saccharomyces cerevisiae encodes an N-acetyltransferase whose acetylation of the N terminus of the L-A double-stranded RNA virus major coat protein (gag) is necessary for viral assembly. We show that the first 4 amino acids of the L-A gag protein sequence, MLRF, are a portable signal for N-terminal acetylation by MAK3. Amino acids 2, 3, and 4 are each important for acetylation by the MAK3 enzyme. In yeast cells, only three mitochondrial proteins are known to have the MAK3 acetylation signal, suggesting an explanation for the slow growth of mak3 mutants on nonfermentable carbon sources.     

胸腺素α1的乙酰化修饰不依赖于乙酰转移酶RimL

目的：考察大肠杆菌乙酰转移酶RimL对胸腺素α1（Tα1）乙酰化修饰的影响。方法：构建含500bp同源臂的卡那抗性基因打靶片段,利用Red同源重组系统,使大肠杆菌B121（DE3）的rimL基因插入失活,随后导入质粒pCP20去除抗性基因,构建突变菌株rimL-BL21（DE3）;将重组质粒pET-Tα1-L12分别转入出发菌株和突变菌株中进行表达,经固定金属离子亲和层析和反向高效液相层析后,将所得纯品进行质谱分析,精确测定相对分子质量。结果：PCR鉴定结果证明成功敲除rimL基因;质谱结果表明,rimL基因敲除菌中所表达的Tα1-L12融合蛋白与出发菌株一样,均有部分乙酰化修饰。结论：Tα1的乙酰化修饰并不依赖于RimL。     

The RimL Transacetylase Provides Resistance to Translation Inhibitor Microcin C

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.     

Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18

The three ribosomal proteins L7, S5, and S18 are included in the rare subset of prokaryotic proteins that are known to be N(alpha)-acetylated. The GCN5-related N-acetyltransferase (GNAT) protein RimI, responsible for the N(alpha)-acetylation of the ribosomal protein S18, was cloned from Salmonella typhimurium LT2 (RimI(ST)), overexpressed, and purified to homogeneity. Steady-state kinetic parameters for RimI(ST) were determined for AcCoA and a peptide substrate consisting of the first six amino acids of the target protein S18. The crystal structure of RimI(ST) was determined in complex with CoA, AcCoA, and a CoA-S-acetyl-ARYFRR bisubstrate inhibitor. The structures are consistent with a direct nucleophilic addition-elimination mechanism with Glu103 and Tyr115 acting as the catalytic base and acid, respectively. The RimI(ST)-bisubstrate complex suggests that several residues change conformation upon interacting with the N terminus of S18, including Glu103, the proposed active site base, facilitating proton exchange and catalysis.     

Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.     

Identification of the human N(alpha)-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression

Protein N(alpha)-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein N(alpha)-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific N(alpha)-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G(0)/G(1)-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G(0)/G(1)-phase and increased the fraction of cells in the sub-G(0)/G(1)-phase. In summary, we show for the first time a vertebrate NatB protein N(alpha)-acetyltransferase complex essential for normal cell proliferation.     

Structure and functions of the GNAT superfamily of acetyltransferases

The Gcn5-related N-acetyltransferases are an enormous superfamily of enzymes that are universally distributed in nature and that use acyl-CoAs to acylate their cognate substrates. In this review, we will examine those members of this superfamily that have been both structurally and mechanistically characterized. These include aminoglycoside N-acetyltransferases, serotonin N-acetyltransferase, glucosamine-6-phosphate N-acetyltransferase, the histone acetyltransferases, mycothiol synthase, protein N-myristoyltransferase, and the Fem family of amino acyl transferases.     

Dependence of ORC silencing function on NatA-mediated Nalpha acetylation in Saccharomyces cerevisiae

N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was N(alpha) acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.     

Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.     

Surprising arginine biosynthesis: a reappraisal of the enzymology and evolution of the pathway in microorganisms.

Major aspects of the pathway of de novo arginine biosynthesis via acetylated intermediates in microorganisms must be revised in light of recent enzymatic and genomic investigations. The enzyme N-acetylglutamate synthase (NAGS), which used to be considered responsible for the first committed step of the pathway, is present in a limited number of bacterial phyla only and is absent from Archaea. In many Bacteria, shorter proteins related to the Gcn5-related N-acetyltransferase family appear to acetylate l-glutamate; some are clearly similar to the C-terminal, acetyl-coenzyme A (CoA) binding domain of classical NAGS, while others are more distantly related. Short NAGSs can be single gene products, as in Mycobacterium spp. and Thermus spp., or fused to the enzyme catalyzing the last step of the pathway (argininosuccinase), as in members of the Alteromonas-Vibrio group. How these proteins bind glutamate remains to be determined. In some Bacteria, a bifunctional ornithine acetyltransferase (i.e., using both acetylornithine and acetyl-CoA as donors of the acetyl group) accounts for glutamate acetylation. In many Archaea, the enzyme responsible for glutamate acetylation remains elusive, but possible connections with a novel lysine biosynthetic pathway arose recently from genomic investigations. In some Proteobacteria (notably Xanthomonadaceae) and Bacteroidetes, the carbamoylation step of the pathway appears to involve N-acetylornithine or N-succinylornithine rather than ornithine. The product N-acetylcitrulline is deacetylated by an enzyme that is also involved in the provision of ornithine from acetylornithine; this is an important metabolic function, as ornithine itself can become essential as a source of other metabolites. This review insists on the biochemical and evolutionary implications of these findings.     

N-terminal acetylation of ectopic recombinant proteins in Escherichia coli

N-terminal acetylation is a protein modification common in eukaryotes, but rare in prokaryotes. Here, we characterized five mammalian stathmin-like subdomains expressed in Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoESI Q-TOF tandem mass spectrometry. We revealed that RB3(SLD) and RB3'(SLD) are N(alpha)-acetylated, whereas SCG10(SLD) and SCLIP(SLD), although identical up to residue 6, are not, as well as stathmin. To assess the influence of the N-terminal sequences on N(alpha)-acetylation, we exchanged residues 7 and 8 between acetylated RB3(SLD) and unacetylated SCG10(SLD), and showed that it reversed the acetylation pattern. Our results demonstrate that ectopic recombinant proteins can be extensively N(alpha)-acetylated in E. coli, and that the rules governing N(alpha)-acetylation are complex and involve the N-terminal region, as in eukaryotes.