A Single Enzyme Transforms a Carboxylic Acid into a Nitrile through an Amide Intermediate

Article abstract of DOI:10.1002/anie.201504505

The biosynthesis of nitriles is known to occur through specialized pathways involving multiple enzymes; however, in bacterial and archeal biosynthesis of 7-deazapurines, a single enzyme, ToyM, catalyzes the conversion of the carboxylic acid containing 7-carboxy-7-deazaguanine (CDG) into its corresponding nitrile, 7-cyano-7-deazaguanine (preQ₀). The mechanism of this unusual direct transformation was shown to proceed via the adenylation of CDG, which activates it to form the newly discovered amide intermediate 7-amido-7-deazaguanine (ADG). This is subsequently dehydrated to form the nitrile in a process that consumes a second equivalent of ATP. The authentic amide intermediate is shown to be chemically and kinetically competent. The ability of ToyM to activate two different substrates, an acid and an amide, accounts for this unprecedented one-enzyme catalysis of nitrile synthesis, and the differential rates of these two half reactions suggest that this catalytic ability is derived from an amide synthetase that gained a new function. Double duty: In bacterial and archeal biosynthesis of 7-deazpurines, a single enzyme, ToyM, catalyzes the two-step conversion of the carboxylic acid 7- carboxy-7-deazaguanine (CDG) into the corresponding nitrile, 7-cyano-7-deazaguanine (preQ₀). The mechanism of this unusual direct transformation proceeds via the adenylation of CDG. This activates it to form the amide intermediate 7-amido-7-deazaguanine (ADG), which is in turn activated to form the nitrile.

Full text of DOI:10.1002/anie.201504505

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Chemie
International Edition: DOI: 10.1002/anie.201504505
German Edition: DOI: 10.1002/ange.201504505
Biosynthesis
Hot Paper
A Single Enzyme Transforms a Carboxylic Acid into a Nitrile through
an Amide Intermediate
Micah T. Nelp and Vahe Bandarian*
Abstract: The biosynthesis of nitriles is known to occur
through specialized pathways involving multiple enzymes;
however, in bacterial and archeal biosynthesis of 7-deazapur-
ines, a single enzyme, ToyM, catalyzes the conversion of the
carboxylic acid containing 7-carboxy-7-deazaguanine (CDG)
into its corresponding nitrile, 7-cyano-7-deazaguanine
(preQ0). The mechanism of this unusual direct transformation
was shown to proceed via the adenylation of CDG, which
activates it to form the newly discovered amide intermediate 7-
amido-7-deazaguanine (ADG). This is subsequently dehy-
drated to form the nitrile in a process that consumes a second
equivalent of ATP. The authentic amide intermediate is shown
to be chemically and kinetically competent. The ability of
ToyM to activate two different substrates, an acid and an
amide, accounts for this unprecedented one-enzyme catalysis
of nitrile synthesis, and the differential rates of these two half
reactions suggest that this catalytic ability is derived from an
amide synthetase that gained a new function.
Figure 1. Two biosynthetic pathways to nitrile secondary moieties in
N
itrile-containing products are widespread in nature and
nature.
are found in secondary metabolites such as cyanoglucosides
and alkanenitriles.[1] Although these are increasingly being
recognized as important next-generation antibiotics and
cytotoxic agents with pharmaceutical potential,[2,3] the bio-
synthesis of these compounds is very poorly understood. Only
one biosynthetic route, which utilizes at least three separate
enzymes, has been described in mechanistic detail, and it
involves aldoxime formation[4] and a subsequent dehydra-
tion[5,6] (Figure 1). The discovery of the 7-deazapurine bio-
synthetic operon of Streptomyces rimosus revealed the first
enzyme capable of acting on a carboxylic acid to form
a nitrile[7] (Figure 1). This nitrile synthetase, ToyM, was shown
to be ATP dependent and to use ammonia as the source of the
nitrile nitrogen.[8] Homologues of ToyM are widely distrib-
uted since this nitrilation is essential to the biosynthesis of the
modified nucleosides archaeosine and the ubiquitous queuo-
sine, as well as many pyrrolopyrimidine nucleoside anti-
biotics, including toyocamycin and sangivamycin.[9]
amine nitrogens.[10,11] The crystal structure of ToyM, solved
before its enzymatic function was known, showed the
presence of a structural zinc divalent cation and revealed
that it adopts a PPi loop structure characteristic of ATP-
utilizing enzymes.[12] This suggests that the mechanistic
strategy employed by ToyM may involve adenylation. The
simplest mechanism for ToyM (Figure 2) would be adenyla-
tion to activate 7-carboxy-7-deazaguanine (CDG), followed
by the addition of ammonia to generate 7-amido-7-deaza-
guanine (ADG). Activation of ADG by a second equivalent
of ATP would precede collapse to form the nitrile product 7-
cyano-7-deazaguanine (preQ0). Such a mechanism would be
a rare example of a single enzyme activating two functional
ToyM bears evolutionary similarity to glutamine-depen-
dent asparagine synthetases and the lysidine-forming enzyme
TilS, both of which employ ATP in the adenylation of
substrate molecules, thus poising them for condensation with
[*] M. T. Nelp, Prof. V. Bandarian
Department of Chemistry and Biochemistry, University of Arizona
1041 East Lowell Street, Biological Sciences West, Tucson
AZ 85721-0088 (USA)
E-mail: vahe@email.arizona.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201504505.
Figure 2. Proposed mechanism of ToyM.
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groups, both the acid as well as the resultant amide
intermediate, and presents a new paradigm for the biosyn-
thesis of nitriles from carboxylic acids.
nesium was required to observe maximal quantities of the
intermediate (Figure S2B in the Supporting Information).
The identity of this intermediate was confirmed by
comparison with the UV/Vis spectra and mass spectra of
authentic ADG, which was synthesized enzymatically from
preQ0 by using ToyJKL, a nitrile hydratase from the same
pathway as ToyM.[13] To determine whether ADG is a genuine
intermediate, it was incubated with ToyM and ATP. In these
experiments (Figure 3B), preQ0 was formed at the expense of
ADG with nearly identical t1/2 values (ca. 4 min). In addition,
AMP formed with a profile that nearly mirrored the
formation of preQ0, thus suggesting that adenylation of
ADG is the likely mode of activation. Therefore, the
formation of preQ0 from CDG appears to occur in two
ATP-dependent half reactions, the first of which leads to the
formation of ADG, which is converted into preQ0 in the
second step.
Direct evidence of the nature of the activated intermedi-
ate was obtained by combining ToyM, CDG, and ATP in the
absence of ammonia. This reaction was filtered to remove the
enzyme and analyzed by ion pairing LC–MS in positive-ion
mode. In these experiments, we detected the formation of an
intermediate that elutes at ca. 25 min. Examination of the
mass spectrum suggested that the peak appears at m/z
524.1030, which is consistent with adenylated CDG (theoret-
ical m/z 524.1044; Figure 4). The appearance of this new peak
is dependent on the presence of ToyM, CDG, and ATP, but it
is abolished in the presence of ammonia (Figure 4A).
To examine the nitrilation catalyzed by ToyM, the
recombinant protein was produced in an E. coli expression
host. Protein was quantified by amino acid analysis, and
metals analysis confirmed the presence of 1.1 Æ 0.2 equiv-
alents of Zn2+ per monomer. To test the mechanistic proposal
in Figure 2, we incubated ToyM with CDG, ATP, and
magnesium, and monitored the reaction by LC–MS (Fig-
ure S1 in the Supporting Information). We found that
magnesium supplementation significantly increased product
formation (Figure S2A in the Supporting Information), and it
was routinely included in the assays. The HPLC traces showed
that preQ0 was produced as previously shown, as well as an
intermediate that increased as CDG was consumed and
decreased as preQ0 formed (Figure 3A). This intermediate
exhibited a UV/Vis spectrum highly similar to that of CDG
(Figure S3 in the Supporting Information) and a mass con-
sistent with 7-amido-7-deazaguanine (ADG; [M + H]+ m/z of
194.0670; theoretical 194.0678). As with preQ0, added mag-
Figure 4. A) Extracted ion traces from ToyM reactions with CDG and
ATP in the absence of ammonium ions, showing the adenylated
intermediate forms only when all three are present. B) MS/MS analysis
of the adenylated intermediate showing fragments of CDG and AMP.
The identity of adenylated CDG was confirmed by using
U-13C10-labeled ATP. The resulting covalent adduct exhibited
the expected mass shift from m/z 524.1030 to m/z 534.1376
(theoretical m/z 534.1379), which is consistent with the
incorporation of ten 13C atoms from the labeled ATP. MS/
MS analysis of the intermediate further confirmed its identity
by revealing fragments consistent with expulsion of [CDG +
H]+ (m/z 195.0511) and a ketene-like fragment of [CDG]+ (m/
z 177.0406). We also observed fragments corresponding to
[adenosine phosphate + H]+ (m/z 348.0701) and [adenine +
H]+ (m/z 136.0615), both of which shift when 13C-lableled
ATP is used. With the labeled substrate, [13C10-adenosine
phosphate + H]+ is observed at m/z 358.1035 and [13C5-
adenine + H]+ at m/z 141.0782 (Figure 4B and Figure S4 in
Figure 3. A) Reaction of ToyM with CDG, ammonium chloride, and
ATP. B) Reaction of ToyM with ADG and ATP.
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the Supporting Information). We note that the presence of
magnesium abolished the buildup of the adenylated inter-
mediate, presumably as a result of magnesium catalyzing the
hydrolysis of the adenylated intermediate in solution. There-
fore, these assays were all carried out in the absence of added
divalent cation.
To further examine whether the acid and amide substrates
are activated in a similar manner, ToyM was incubated with
ATP, CDG, and ammonia or ADG for 8.5 h, after which these
reaction mixtures were tested by using a coupled enzyme
pyrophosphate detection assay.[14] Reactions with both sub-
strates showed the characteristic signal for the presence of
pyrophosphate in at least the same stoichiometry as that of
the preQ0 product (Figure 5). Taken together with the
formation of AMP with both substrates, this result demon-
strates conclusively that ToyM is capable of catalyzing the
same adenylation reaction on two substrates, CDG and ADG,
to eventually form the nitrile preQ0.
is an elegant example of finding new chemical capability
through the promiscuity of an amide synthetase enzyme to
achieve what requires at least three enzymes in other known
synthetic pathways.
In conclusion, ToyM is an amide synthetase, as well as
a nitrile synthetase. Its ability to accept both the acid and
amide forms of CDG enables sequential amidation and
dehydration to the nitrile. This provides a new paradigm for
studying the origin of the many nitrile-containing secondary
metabolites, the biosynthetic provenance of which is currently
unknown, and it gives molecular detail to the synthesis of an
important intermediate in many pathways, the nitrile-con-
taining compound preQ0.
Keywords: bioorganic chemistry · biosynthesis · enzymes ·
evolution · nitriles
How to cite: Angew. Chem. Int. Ed. 2015, 54, 10627–10629
Angew. Chem. 2015, 127, 10773–10775
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Figure 5. Pyrophosphate-dependent consumption of NADH through
a coupled enzyme assay showing that with both substrates (CDG and
ADG), ToyM produces pyrophosphate, whereas with either no sub-
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Enzyme promiscuity has been integral to the formation of
the immense biosynthetic repertoire found in nature.[15,16]
According to this model, the efficiency of enzymes for their
ancestral primary reaction is likely higher than that for the
reactions for which they gain catalytic ability over time.[17]
ToyM bears tantalizing clues to its evolution in that the first
half reaction, the amidation, proceeds more rapidly than
dehydration to the nitrile in the second half reaction. It is thus
likely that this unprecedented and simplified nitrile synthesis
Received: May 18, 2015
Published online: July 17, 2015
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