N-acetyltransferases from three different organisms displaying distinct selectivity toward hexosamines and N-terminal amine of peptides

Article abstract of DOI:10.1016/j.carres.2018.11.011

N-acetyltransferases are a family of enzymes that catalyze the transfer of the acetyl moiety (–COCH₃) from acetyl coenzyme A (Acetyl-CoA) to a primary amine of acceptor substrates from small molecules such as aminoglycoside to macromolecules of various proteins. In this study, the substrate selectivity of three N-acetyltransferases falling into different phylogenetic groups was probed against a series of hexosamines and synthetic peptides. GlmA from Clostridium acetobutylicum and RmNag from Rhizomucor miehei, which have been defined as glucosamine N-acetyltransferases, were herein demonstrated to be also capable of acetylating the free amino group on the very first glycine residue of peptide in spite of varied catalytic efficiency. The human recombinant N-acetyltransferase of Naa10p, however, prefers primary amine groups in the peptides as opposed to glucosamine. The varied preference of GlmA, RmNag and Naa10p probably arose from the divergent evolution of these N-acetyltransferases. The expanded knowledge of acceptor specificity would as well facilitate the application of these N-acetyltransferases in the acetylation of hexosamines or peptides.

Full text of DOI:10.1016/j.carres.2018.11.011

CarbohydrateResearch472(2019)72–75
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
N-acetyltransferases from three different organisms displaying distinct
selectivity toward hexosamines and N-terminal amine of peptides
Peiru Zhanga,1, Pei Liua,1, Yangyang Xua, Yulu Liangb, Peng George Wanga, Jiansong Chenga,∗
a College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, PR China
b College of Chemistry, Nankai University, Tianjin, 300071, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
N-acetyltransferases are a family of enzymes that catalyze the transfer of the acetyl moiety (eCOCH3) from
acetyl coenzyme A (Acetyl-CoA) to a primary amine of acceptor substrates from small molecules such as ami-
noglycoside to macromolecules of various proteins. In this study, the substrate selectivity of three N-acetyl-
transferases falling into different phylogenetic groups was probed against a series of hexosamines and synthetic
peptides. GlmA from Clostridium acetobutylicum and RmNag from Rhizomucor miehei, which have been defined as
glucosamine N-acetyltransferases, were herein demonstrated to be also capable of acetylating the free amino
group on the very first glycine residue of peptide in spite of varied catalytic efficiency. The human recombinant
N-acetyltransferase of Naa10p, however, prefers primary amine groups in the peptides as opposed to glucosa-
mine. The varied preference of GlmA, RmNag and Naa10p probably arose from the divergent evolution of these
N-acetyltransferases. The expanded knowledge of acceptor specificity would as well facilitate the application of
these N-acetyltransferases in the acetylation of hexosamines or peptides.
N-acetyltransferase
Acetylation
Glucosamine
Peptide
Specificity
1. Introduction
eukaryotes, very few proteins were found to be acetylated in bacteria.
Up to present, only four proteins including RimI, RimJ and RimL from
N-terminal acetylation is one of the most common protein post-
translational modifications in eukaryotes, and about 85% of different
varieties of eukaryotic proteins are acetylated [1,2]. In general, protein
N-terminal acetylation plays a role in various cellular functions in-
cluding stimulation of DNA binding, protein-protein interactions and
protein stability [3,4]. Protein N-terminal acetylation also affects
varieties of normal and pathological cell biology [5,6].
Salmonella enterica and YhY from Escherichia coli with NAT activity have
been reported and are believed to be homologues of eukaryotic NATs
[10,11].
In the present study, two NATs of GlmA and RmNag derived from C.
acetobutylicum and R. miehei, respectively, were unexpectedly found to
be capable of acetylating the free amino group on the very first glycine
residue of peptide in spite of varied catalytic efficiency. GlmA and
RmNag have been defined as glucosamine NATs and clustered into
different phylogenetic groups from human NAT Naa10p. Glucosamine
N-acetyltransferases (EC 2.3.1.3) catalyze the transfer of an acetyl
group from Acetyl-CoA to a glucosamine (GlcN) acceptor generating a
N-acetylglucosamine (GlcNAc). The enzymes participating in amino
sugar metabolism also have been found in plants. Albeit distinct pre-
ferences showed by GlmA and Naa10p toward GlcN and peptide with N-
termini of Gly, RmNag exhibits comparable activity toward them. The
distinct specificity of GlmA, RmNag and Naa10p probably resulted from
the divergent evolution, and meanwhile the extended knowledge of
substrate selectivity of these NATs would also facilitate the application
of them in the acetylation of hexosamines or specific peptides.
N-terminal acetyltransferases (NATs) catalyze the transfer of the
acetyl moiety (eCOCH3) from acetyl coenzyme A (Acetyl-CoA) to a
primary amine of acceptor substrates from small molecules such as
aminoglycoside to macromolecules including various proteins [7,8].
Generally, NATs belong to the GCN-5 related N-acetyltransferases fa-
mily (GNAT), which is a significant protein family and containing more
than 100,000 members in all life kingdoms [7,8]. There is very limited
sequence conservation among members of the GNAT superfamily, in
part, reflecting their capacity to bind and modify a diverse array of
substrates. In eukaryotes, a group of NATs (NatA-F) have been proved
to be involved in protein N-terminal acetylation and differ from each
other in subunit composition, substrate preferences and phenotypes
[1,9]. In contrast with the high abundance of N-acetylated proteins in
∗ Corresponding author.
E-mail address: jiansongcheng@nankai.edu.cn (J. Cheng).
1 The authors contributed to this study equally.
https://doi.org/10.1016/j.carres.2018.11.011
Received 13 October 2018; Received in revised form 18 November 2018; Accepted 21 November 2018
Availableonline22November2018
0008-6215/©2018PublishedbyElsevierLtd.

P. Zhang et al.
CarbohydrateResearch472(2019)72–75
2. Results and discussion
2.1. GlmA, RmNag and Naa10p are distantly related
GlmA, a bacterial glucosamine N-acetyltransferase catalyzing the
transfer of an acetyl moiety from Acetyl-CoA to the primary amino
group of GlcN [12,13], was recently employed by our group for pro-
duction of glycopeptides carrying natural eukaryotic N-glycans. In the
approach, GlcN linked peptides made by an engineered N-glycosyl-
transferase (ApNGTQ469A) were further modified by GlmA to generate
GlcNAcylated peptides [14]. Herein, GlmA was unexpectedly found to
be capable of acetylating the free amino group on the very first glycine
residue of peptide in spite of low efficiency. For better understanding
the selectivity of various N-acetyltransferases, we next selected and
expressed two distantly related enzymes for further study. One of them
is characterized as a glucosamine N-acetyltransferase (RmNag) from
fungi R. miehei, while another is the recombinant the catalytic subunit
(Naa10p) of human N-acetyltransferase NatA complex. RmNag is a bi-
functional protein with a β-N-acetylglucosaminidase domain in the N-
terminal region followed by a glucosamine N-acetyltransferase domain,
and was proposed to be involved in chitin metabolism of fungi [15,16].
Though both GlmA and RmNag were defined as glucosamine N-acet-
yltransferases, they shared only 47% sequence similarity. All three
proteins are distantly related and fall into three different phylogenetic
groups [17] (Fig. 1).
Fig. 2. TLC analyses of acetylation products yielded by GlmA, RmNag (a) and
Naa10p (b). Lane 1: Acetyl-CoA; Lane 2: GlcN; Lane 3: GlcNAc; Lane 4a: re-
action mixture of GlmA; Lane 5a: reaction mixture of RmNag; Lane 4b: reaction
mixture of Naa10p.
ManN. The thin-layer chromatography (TLC) analysis indicated that all
three NATs are capable of acetylating GlcN despite of low efficiency
exhibited by Naa10p (Fig. 2). ManN cannot be accepted by all three
NATs as substrate (Fig. S1). GalN, however, was partially acetylated by
the fungal RmNag after 2 h incubation at 37 °C (Fig. S1).
2.3. Both GlmA and RmNag are active toward peptide starting with Gly
By scanning a penal of peptides starting with 20 various common
amino acids, we found that the peptide containing N-terminal Gly is
able to be exclusively recognized by GlmA and RmNag in spite of varied
acetylation efficiency (Fig. 3). Further investigation by using two sets of
peptides with varied amino acids at next two positions (+2 or +3)
following the N-terminal Gly (+1) indicated that negative charged
residues are disliked by both enzymes at either +2 or +3 position. In
contrast, eukaryotic proteins with Ser, Thr and Ala termini are most
frequently acetylated by NatA, and these residues, along with Met and
Gly account for over 95% of the N-terminal acetylated residues. Be-
sides, acidic residues, Asp or Glu, at the succeeding position are pre-
ferred for NatA [9]. Since blast search revealed the presence of NatA
ortholog in R. miehei (Fig. S2), it remained unclear whether the NatA
ortholog and RmNag might play roles in a collaboration manner.
Generally, the selectivity of both GlmA and RmNag at position +3
is more flexible than that at position +2. In comparison with GlmA,
RmNag exhibited more efficient activity and flexible selectivity toward
peptides (Fig. 4).
2.2. All three N-acetyltransferases are capable of acetylating GlcN
The specificity of GlmA, RmNag and Naa10p toward hexosamines
was probed by using three monosaccharides including GlcN, GalN and
The thin-layer chromatography (TLC) analysis showed that both
GlmA and RmNag are able to efficiently modify GlcN (Fig. 2a).
Fig. 1. Phylogenetic tree of three N-acetyltransferases. Neighbor-Joining (NJ)
method was used to create this unrooted tree. Bootstrap values (expressed as
percentages of 1000 replications) greater than 50% are indicated above the
branches. The protein identification numbers are followed by species in-
formation.
Fig. 3. The peptide GGLSFV starting with glycine can be acetylated by RmNag
(a) and GlmA (b), generating Ac-GGLSFV.
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CarbohydrateResearch472(2019)72–75
more roles in cell than previously proposed (involvement in chitin
metabolism of fungi). On the other hand, the preference of GlmA to-
ward GlcN make it a better catalyst for producing GlcNAcylated pep-
tides or proteins. PA3944, a Gcn5-related acetyltransferase derived
from Pseudomonas aeruginosa, has been proven to accept both glycine
and GlcN and exhibits around 3-fold efficiency for the former [19].
2.4. Naa10p is a N-acetyltransferase for specific peptides
Naa10p is the catalytic subunit of the N-terminal acetyltransferase
NatA and can independently acetylate specific peptides or proteins
[20,21]. It is reported that Naa10p appears to favor peptides starting
with acidic residues, such as Glu-starting N-termini [22,23]. Herein, the
assays containing different monosaccharides (GlcN, GalN or ManN)
indicated that Naa10p can also acetylate GlcN generating N-acet-
ylglucosamine (Fig. 2b), while the acetylation efficiency is relatively
low in comparison with that catalyzed by the bacterial GlmA and the
fungal RmNag. The preference of Naa10p toward GlcN and peptide was
also explored by using a pair of substrates Ac-GGAN(GlcN)LTYTIE and
EGLSFV with acidic N-termini. As shown in Fig. 5, peptide with the
acidic the N-termini is much more preferred by Naa10p in comparison
with GlcN.
3. Conclusions
Taken together, the selectivity of three distantly related NATs was
comprehensively explored and compared. GlmA from C. acetobutylicum
is able to exclusively acetylate GlcN instead of GalN and ManN, and
exhibits much more efficiency toward GlcN as opposed to peptide. In
contrast, the human Naa10p preferred peptide with acidic N-termini to
GlcN. Therefore, GlmA and Naa10p are appropriate to be defined as
glucosamine N-acetyltransferase and peptide/protein N-terminal acet-
yltransferase, respectively, as previously reported. However, RmNag
from R. miehei showed matched acetylation activity against both GlcN
and peptide with N-terminal Gly, indicating that RmNag may play more
roles in cell than previously proposed (involvement in chitin metabo-
lism of fungi). NATs diverge from other members of the GNAT super-
family and differ from each other in the type of substrate N-termini
preferred. The systematic comparison of their specificity would facil-
itate selecting suitable catalyst in acetylation of various molecules
(GlcN or peptide/protein). For example, the yeast NatB complex has
recently been transferred to E. coli for exclusively acetylating α-synu-
clein carrying a Met-Asp-N-termini [24].
Fig. 4. The acetylation conversion rate of GlmA and RmNag toward a penal of
peptides bearing varied residues at +2 position (a) or +3 position (b). For each
position, representative amino acids containing basic, acidic, hydroxyl, small
hydrophobic, or aromatic side chains were evaluated.
4. Experimental
4.1. Materials
Acetyl-CoA was purchased from Sigma-Aldrich (St.Louis, MO, USA).
Glucosamine (GlcN), galactosamine (GalN), and mannosamine (ManN)
were purchased from Aladdin (Shanghai, China). Peptides were syn-
thesized as previously described [18]. GlcN-peptide was synthesized by
an engineered N-glycosyltransferase (ApNGTQ469A) as previously de-
scribed [14].
Fig. 5. The acetylation conversion rate of GlmA, RmNag and Naa10p toward
GGANLTYTIE and Ac-GGAN(GlcN)LTYTIE, as well as EGLSFV (preferred by
Naa10p). All acetylation assays were duplicated, performed at 37 °C for 2 h and
analyzed by RP-HPLC. Acetylated products were further identified by MALDI-
TOF-MS (Figs. S3–S6).
4.2. Cloning, expression and purification of recombinant GlmA, RmNag
and human Naa10p
Therefore, to compare the efficiency of GlmA and RmNag toward GlcN
moiety and Gly-starting peptide, a GlcN-linked Ac-peptide (Ac-GGAN
(GlcN)LTYTIE) where the GlcN was transferred to Asn residue of the N-
glycosylation sequon (Asn-Leu-Thr) by an engineered N-glycosyl-
transferase ApNGTQ469A [18], and an unmodified peptide with same
amino acid sequence (GGANLTYTIE) were used as acceptors and as-
sayed. As shown in Fig. 5, the amine group of GlcN is much more
preferred by GlmA in comparison with that of the N-terminal Gly,
whereas RmNag showed almost equal activity against both acceptor
substrates. The more flexible acceptor selectivity of RmNag might imply
Full-length synthetic genes (codon optimized for the expression
system of Escherichia coli) of glmA (GenBank: AE001437) and rmNag
(GenBank: KC357713, the Asp268 was substituted by Ala) were syn-
thesized by Genewiz (Suzhou, China) and then cloned into expression
vector pET22b(+) and pET28a, respectively. The synthetic gene of
naa10p (GenBank: KJ892483) was cloned into pMAL-c2x as a N-term-
inal maltose-binding protein (MBP)-fused and His6-tagged recombinant
protein. The expression strain E. coli BL21 (DE3) harboring the
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CarbohydrateResearch472(2019)72–75
corresponding plasmids were cultured in LB medium (50 μg/mL ampi-
cillin for GlmA and Naa10p, or 50 μg/mL kanamycin for RmNag) with
vigorous sharking. When the optical density at 600 nm (OD600) reached
0.8, 0.1 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) was
added and then incubated at 18 °C for 20 h.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.carres.2018.11.011.
Cells were harvested by centrifugation (5000×g, 4 °C, 20 min), re-
suspended in lysis buffer (100 mM Tris-HCl, 5 mM NaCl, pH 8.0), and
then broken by sonication. After centrifugation (18,000×g, 4 °C,
30 min) of the cell lysate, the supernatant was loaded onto a Ni2+-NTA
affinity column (Qiagen, Hilden, Germany). Before the lysate was
loaded, the column was pre-equilibrated with 10 column volumes of
binding buffer (10 mM imidazole, 0.5 M NaCl and 20 mM Tris-HCl, pH
8.0). After washing with 10 column volumes of binding buffer and
washing buffer (30 mM imidazole, 0.5 M NaCl and 20 mM Tris-HCl, pH
8.0), the target proteins were eluted with elution buffer (250 mM imi-
dazole, 0.5 M NaCl and 20 mM Tris-HCl, pH 8.0). Elution fractions were
analyzed by 12% SDS-PAGE with Coomassie staining. The concentra-
tions of purified enzymes were obtained using a BCA Protein Assay Kit
(Pierce Biotechnology, Rockford, IL) with bovine serum albumin as a
protein standard.
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Financial support for this work was provided by the National
Science Foundation grants: 21372130 and 81302682.
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