NosA catalyzing carboxyl-terminal amide formation in nosiheptide maturation via an enamine dealkylation on the serine-extended precursor peptide

Article abstract of DOI:10.1021/ja106571g

The carboxyl-terminal amide group has been often found in many bioactive peptide natural products, including nosiheptide belonging to the over 80 entity-containing thiopeptide family. Upon functional characterization of a novel protein NosA in nosiheptide biosynthesis, herein we report an unusual C-terminal amide forming strategy in general for maturating certain amide-terminated thiopeptides by processing their precursor peptides featuring a serine extension. NosA acts on an intermediate bearing a bis-dehydroalanine tail and catalyzes an enamide dealkylation to remove the acrylate unit originating from the extended serine residue.

Full text of DOI:10.1021/ja106571g

Published on Web 11/03/2010
NosA Catalyzing Carboxyl-Terminal Amide Formation in Nosiheptide
Maturation via an Enamine Dealkylation on the Serine-Extended Precursor
Peptide
Yi Yu,† Heng Guo,† Qi Zhang,† Lian Duan,† Ying Ding,† Rijing Liao,† Chun Lei,† Ben Shen,‡ and
Wen Liu*,†
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China, and DiVision of Pharmaceutical
Sciences, School of Pharmacy, UniVersity of Wisconsin, Madison, Wisconsin 53705, United States
Received July 24, 2010; E-mail: wliu@mail.sioc.ac.cn
Scheme 1. Variable Mechanisms for Carboxyl-Terminal Amide
Formation
Abstract: The carboxyl-terminal amide group has been often
found in many bioactive peptide natural products, including
nosiheptide belonging to the over 80 entity-containing thiopeptide
family. Upon functional characterization of a novel protein NosA
in nosiheptide biosynthesis, herein we report an unusual C-
terminal amide forming strategy in general for maturating certain
amide-terminated thiopeptides by processing their precursor
peptides featuring a serine extension. NosA acts on an intermedi-
ate bearing a bis-dehydroalanine tail and catalyzes an enamide
dealkylation to remove the acrylate unit originating from the
extended serine residue.
The amidation catalyzed by asparagine synthetase-like proteins
usually utilizes the γ-carboxamide of Gln to in situ generate a
nascent ammonia, attack of which to the activated carboxylate
results in amide formation with Glu, AMP, and PPi as the
coproducts (Scheme 1A).1 For many peptide natural products,
carboxyl-terminal amides can be generated endogenously (Scheme
1B), as shown in the biosynthesis of ribosomally synthesized animal
hormones and nonribosomally microbial metabolites such as
melithiazols.2 Their amide moieties arise from the oxidative
cleavage of C-terminal Gly-extended peptides accompanied by
glyoxylate production,3 during which the R-hydroxylation of Gly
followed by dealkylation is divalent ion or flavin dependent. Distinct
from these well established strategies, we here report a new way
to furnish the C-terminal amide in general for maturating certain
thiopeptides, based on in ViVo and in Vitro characterization of a
novel enzyme NosA that acts on the Ser-extended precursor peptide
in nosiheptide (1) biosynthesis.
involved in the late-stage tailoring to give 1.5 Whether or not NosA
catalyzes C-terminal amide formation remains elusive, given that
all NosA homologues in the database are annotated as unknown
proteins.
We first validated the role of NosA in 1 maturation by gene
inactivation in the producing strain Streptomyces actuosus. To
exclude the potential effects on downstream gene expression, nosA
mutant strain SL4008 was generated by in-frame deletion. SL4008
completely lost the ability to produce 1; however, it produced a
distinct compound (2) that exhibits the UV absorption pattern quite
similar to that of 1 (Figure 2, II). In trans expression of nosA in
SL4008 led to the complete conversion of 2 to 1 in the resulting
recombinant strain SL4010 (Figure 2, III), confirming the indis-
pensability of nosA in 1 biosynthesis. For structural elucidation, 2
was purified and then subjected to spectroscopic analysis (Figures
Nosiheptide is one of the parent compounds in the thiopeptide
family that contains over 80 clinically interesting entities.4 The
characteristic macrocyclic core of 1 consists of a hydroxypyridine
central to multiple thiazoles, dehydroamino acids, and an amide-
terminated peptide chain (Figure 1). We have recently unveiled
that 1 has a ribosomal origin as other members newly to be
characterized biosynthetically,5,6 showing complex posttranslational
modifications on the precursor peptide NosM. The structural peptide
of NosM, consistent with the amino acids constituting the peptide
backbone, possesses an extended Ser residue proven to be the
nitrogen source of the terminal amide.7 Functional assignment of
biosynthetic genes in the nos cluster allows for identifying a
hypothetic gene, nosA, which encodes a 151-aa protein likely
† Shanghai Institute of Organic Chemistry.
‡ University of Wisconsin.
Figure 1. Precursor peptide NosM and structures of 1 and 2. The structural
peptide is shown in bold. Asterisk indicates the extended Ser.
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C O M M U N I C A T I O N S
chemical conversion. NosA was overproduced in Escherichia coli
BL21(DE3) in a 6 × His tagged form and then purified to
homogeneity for in Vitro studies (Figure S6A). The resulting protein
was subjected to MALDI-TOF MS analysis, exhibiting an M+ ion
at m/z ) 18 302.405 (Figure S6B). Taking the calculated molecular
weight 18 318.46 and UV-absorptions into account, it is unlikely
that any cofactor binds to NosA. In the presence of NosA, the
conversion of 2 to 1 efficiently took place (Figure 2, VII), requiring
no cofactor. To probe the catalytic chemistry, 2,4-dinitrophenyl-
hydrazine was added in the NosA-catalyzed reaction for derivati-
zation, leading to identification of the coproduct pyruvate (3,
corresponding to the derivative 2-(2,4-dinitrophenylhydrazono)-
propanoic acid) (Figure S7). These results validated that NosA acts
on 2, catalyzing the amide moiety formation via a CR-N bond
cleavage to afford 1 by eliminating the terminal acrylate moiety.
The characteristic thiopeptide framework might be necessary for
substrate recognition, since 2-acetamidoacrylic acid, a mimic to
the peptidyl tail of 2, cannot be turned over by NosA to generate
3.
For analyzing the NosA-catalyzed reaction in Vitro, we evaluated
the stability of the substrate 2 before the condition optimization. 2
was stable at pH from 3.0 to 8.0 (Figure S8). By contrast, the
incubation at pH 9.0 resulted in a time-dependent transformation
of 2 to a major distinct compound (X, HR-ESI-MS m/z [M + H]+
calcd 1310.1717 for C54H48N13O15S6, found 1310.1765), which, with
the 18-Da increase in molecular weight, could be a hydrated or
side-ring-hydrolyzed product of 2 (Figure 2, IX, and Figure S9).
X can be efficiently converted by NosA to Y (HR-ESI-MS m/z [M
+ H]+ calcd 1240.1662 for C51H46N13O13S6, found 1240.1725), a
putative acrylate-removed analogue (Figure 2, X), excluding the
possibility of H2O addition onto the terminal dehydroalanine unit.
The mixtures containing X and Y, respectively, were therefore
subjected to sulfhydryl derivatization by addition of 5,5′-dithio-
bis(2-nitrobenzoic acid) (DTNB) (Figure S10), showing the genera-
tion of their corresponding derivatives X′ (ESI-MS m/z [M + H]+
calcd 1507.1, found 1506.6) and Y′ (ESI-MS m/z [M + H]+ calcd
1437.1, found 1436.8). This indicated the cleavage of the thioester
bond by hydrolysis at high pH to give a free sulfhydryl group.
Further, MS/MS analysis supported the deduced structures of X
and Y (Figures S11 and S12), both of which are indole side-ring-
opened. The finding that NosA tolerates X as the substrate is
consistent with the hypothesis that the main thiopeptide core plays
a key structural role in the enzyme action, as a similar modification
for maturation could be involved in the biosynthesis of the
monocyclic thiopeptide GE2270A.6d
Figure 2. Validation of NosA as a terminal amide-forming protein. In ViVo
1 or 2 production in S. actuosus strains, including the wild type (I); nosA
mutant strain SL4008 (II); SL4010, a SL4008 derivative carrying nosA (III);
and SL4009, a SL4008 derivative carrying nocA (IV). Using standard 1
(V) and purified 2 (VI) as the controls, in Vitro conversion of 2 to 1 catalyzed
by active NosA (VII) and NosA inactivated by heating (VIII); transformation
of 2 to X by incubation at pH 9.0 for 12 h in the absence of NosA (IX);
and transformation of the mixture of 2 and X respectively to 1 and Y in
the presence of NosA (X).
S1-S3). HR-ESI-MS analysis established the molecular formula
as C54H45N13O14S6Na (m/z [M + Na]+ 1314.1467, 1314.1431
calculated), and extensive MS/MS analyses suggested the only
difference of 2 from 1 in the C-terminal functionality of the peptidyl
1
side chain. The H, 13C, and 2D NMR spectra (including HSQC
and HMBC correlations) finally established that 2 is a new
biosynthetic intermediate of 1 that bears a bis-dehydroalanine tail
(Figure 1). These findings strongly supported the involvement of
nosA in amide formation by removing the terminal acrylate unit of
2 to give 1.
Thus, the pH dependence of NosA-catalyzed conversion was
investigated by using 2 as the substrate in the reaction with buffers
varying from 4.0 to 8.5, displaying an optimal activity at pH 8.0
(Figure S13). The addition of EDTA into the reaction mixture did
not afford a significant change in enzymatic activity, suggesting
the metal ion independence of NosA. Under the optimized
conditions, the steady-state kinetic parameters were measured,
showing a conversion with a km value of 93.7 ( 0.7 µM for 2 and
a kcat value of 1200 ( 30 min-1 (Figure S14).
We thereby proposed that the NosA-catalyzed reaction may
proceed via an enamide dealkylation: (1) specific tautomerization
of the terminal dehydroalanine unit of 2 to the corresponding methyl
imine is followed by nucleophilic attack of H2O to generate the
terminally hydrated intermediate, and (2) subsequent cleavage of
the CR-N bond yields 1 with pyruvate production (Scheme 1C).
Consistent with previous studies in chemical synthesis of thios-
trepton,8 the reaction can be base-initiated, whereas acidic condi-
tions would make tautomerization of enamide to the labile imine
The counterparts of nosA were also found for biosynthesizing
some amidated thiopeptides (Figures S4 and S5), as nocA (64%
identity) for nocathiacin and tpdK (34% identity) for GE2270A.6d,f
Similar to NosM in 1 biosynthesis, their precursor peptides contain
one (Ser for NocM) or two (Ser-Ala for TpdA) extended amino
acid(s) at the C-terminus in addition to the sequence for forming
the thiopeptide backbone. We therefore carried out heterologous
complementation to examine if the CR-N cleavage on the
dehydroalanine unit (derived from Ser) is common for maturating
these thiopeptides. nocA was introduced into SL4008, yielding the
recombinant strain SL4009 for product detection. Remarkably, about
50% of 2 was converted to 1 (Figure 2, IV), ascertaining the
functional identity of nocA to nosA. This interchangeability indicates
a strategy in general to afford a C-terminal amide by processing
precursor peptides that feature a Ser residue extension.
To characterize NosA as a novel protein for C-terminal amide
formation, we next explored its cofactor dependence and subsequent
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C O M M U N I C A T I O N S
difficult and lead to a dramatic decrease in NosA activity (Figure
S13). The NosA chemistry is nonoxidative and takes advantage of
the terminal enamide moiety generated from the extended Ser by
the dehydratase pair NosE and NosD that act at the early stage
posttranslational modifications on the precursor peptide NosM.6
This strategy is clearly distinct from that for maturation of the
thiopeptides originating from the non-Ser-extended precursor pep-
Supporting Information Available: Experimental procedures for
gene inactivation and complementation, characterization of reactions
and compounds, protein expression and analysis, sequence analysis,
and bioassay; supplementary tables; and supplementary figures. These
materials are available free of charge via the Internet at http://
pubs.acs.org.
tides, such as thiostrepton and siomycin (Figure S5),7 which
b-d
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be common for maturating the thiopeptides nosiheptide, nocathiacin,
and GE2270A, by processing the Ser-extended precursor peptide
to furnish the terminal amide moiety with pyruvate production. The
NosA chemistry is mechanistically distinct from known C-terminal
amide forming proteins. Taking advantage of the substrate tolerance,
it can be exploited by combinatorial biosynthesis for structural
diversity in thiopeptide natural products.
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