Novel Bisubstrate Inhibitors for Protein N-Terminal Acetyltransferase D.

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitors are competitive to the peptide substrate and noncompetitive to the cofactor, suggesting that NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific toward NatD, displaying about 1000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegypti.

Mosquitoes transmit a number of diseases in animals and humans, including Dengue, Chikungunya and Zika viruses that affect millions of people each year. Controlling the disease-transmitting mosquitoes has proven to be a successful strategy to reduce the viruses transmission. Polyamines are required for the life cycle of the RNA viruses, Chikungunya virus and Zika virus, and a depletion of spermidine and spermine in the host via induction of spermine N-acetyltransferase restricts their replication. Spermine N-acetyltransferase is a key catabolic enzyme in the polyamine pathway, however there is no information of the enzyme identification in any insects. Aliphatic polyamines play a fundamental role in tissue growth and development in organisms. They are acetylated by spermidine/spermine N1-acetyltransferase (SAT). In this study we provided a molecular and biochemical identification of SAT from Aedes aegypti mosquitoes. Screening of purified recombinant proteins against polyamines established that aaNAT5b, named previously based on sequence similarity with identified aaNAT1 in insects, is active to spermine and spermidine. A crystal structure was determined and used in molecular docking in this study. Key residues were identified to be involved in spermine binding using molecular docking and simulation. In addition, SAT transcript was down regulated by blood feeding using a real time PCR test. Based on its substrate profile and transcriptional levels after blood feeding, together with previous reports for polyamines required in arboviruses replication, SAT might be potentially used as a target to control arboviruses with human interference.

N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.

Cotranslational N-terminal (Nt-) acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently, there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study, we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala, or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs.

The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD.

N-terminal acetylation is among the most common protein modifications in eukaryotes and is mediated by evolutionarily conserved N-terminal acetyltransferases (NATs). NatD is among the most selective NATs; its only known substrates are histones H4 and H2A, containing the N-terminal sequence SGRGK in humans. Here we characterize the molecular basis for substrate-specific acetylation by NatD by reporting its crystal structure bound to cognate substrates and performing related biochemical studies. A novel N-terminal segment wraps around the catalytic core domain to make stabilizing interactions, and the α1-α2 and ß6-ß7 loops adopt novel conformations to properly orient the histone N termini in the binding site. Ser1 and Arg3 of the histone make extensive contacts to highly conserved NatD residues in the substrate binding pocket, and flanking glycine residues also appear to contribute to substrate-specific binding by NatD, together defining a Ser-Gly-Arg-Gly recognition sequence. These studies have implications for understanding substrate-specific acetylation by NAT enzymes.