Human Naa50p (Nat5/San) Displays Both Protein Nα- and Nϵ-Acetyltransferase Activity

Abstract

Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine N ⑀ -acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. N ␣ -terminal acetylation occurs on the ribosome where the ␣ amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an N ␣ -acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.

Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

“…The crude protein extract was cleared by centrifugation at 28,000g at 4°C for 20 min and directly used in the enzymatic test system. Activity of crude protein extract of HIS-MBP-NAA10 or HIS-MBP-NAA20 (30 µg for total protein) was tested according to Evjenth et al (2009) for 60 min at 37°C in acetylation buffer (50 mM TrisHCl, pH 7.5, 10% glycerol, 10 mM DTT, and 1 mM EDTA) containing 500 mM [ 3 H] acetyl-CoA (7.48 GBq/mmol; Hartmann Analytics) and 0.2 mM peptide (Genecust). Peptide sequences are as follows: OAS-TL A, ASRIAKDVTERWGRPVGRRRRPVRVYP; SNC1 M, MDTSKDDDMERW-GRPVGRRRRPVRVYP; SNC1 MM, MMDTSKDDDMERWGRPVGRR-RRPVRVYP; SNC1 MAM, MAMDTSKDDDMERWGRPVGRRRRPVRVYP; and Neg CTRL, SPTPPLFSLPRWGRPVGRRRRPVRVYP.…”

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

“…NatA, which harbors the most diversity for substrate selection, is responsible for modifying the majority of all amino-terminally acetylated proteins, which it accomplishes by recognizing proteins with amino-terminal Ala-, Cys-, Gly-, Ser-, Thr-, or Val-residues (5). NatE/NAA50 has only one known biologically relevant substrate that contains the aminoterminal sequence Met-Leu-Gly-Pro, of which only the aminoterminal Met-is absolutely required for catalysis (14). These structures and their accompanying biochemical characterization have provided significant insight into the mechanisms of substrate specificity and catalysis used by NAT enzymes.…”

“…Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6-11). Three additional human NAT enzymes, NatD-NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12)(13)(14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and human NatE, which is the independently active NAA50 enzyme (15, 16). NatA, which harbors the most diversity for substrate selection, is responsible for modifying the majority of all amino-terminally acetylated proteins, which it accomplishes by recognizing proteins with amino-terminal Ala-, Cys-, Gly-, Ser-, Thr-, or Val-residues (5).…”

“…Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6-11). Three additional human NAT enzymes, NatD-NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12)(13)(14).…”

The torments of the cohesin ring

“…In addition, human Ard1 was found to translocate into the nucleus. 45 However, the reports on the ability of N-terminal acetyltransferases to modify internal lysines [46][47][48][49] remain controversial, 50,51 and our attempts to acetylate Smc proteins in vitro using recombinant Nat3/Mdm20 and Ard1/Nat1 N-terminal acetyltransferases were unsuccessful. It is conceivable that it would be necessary to couple an acetylation assay to an in vitro translation system.…”