Journal of the American Chemical Society
COMMUNICATION
experimental evidence that the biosynthesis of pacidamycin
nucleoside 1 starts from a different precursor than for liposido-
mycin-type nucleoside 2 or nikkomycin-related nucleoside 3.
The reaction of Pac13 with uridine did not yield any product
alignments. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(
Figure S21), which strongly suggests that Pac11 is the first
Corresponding Author
r.goss@uea.ac.uk.
enzyme to act in aminonucleoside biosynthesis. The reaction of
Pac11 in combination with Pac13 generated 10 as the major
product from uridine, and uridyl aldehyde 9 was detected as a
minor product (Figure 3 and Figure S22). Enzyme assays using
Pac5 were performed in the presence of PLP and alanine (see
section 4.3 in the SI). As expected, aminonucleoside 7 was
obtained when uridine was incubated with Pac11 and Pac5.
When all three enzymes were incubated together, a new peak
appeared as the major product. Though under the conditions of
the HPLC its retention time was similar to those of 9 and 10, in
LCꢀMS it differed (Figure 3 and Figure S24). Its molecular ion
and MS/MS fragmentation pattern were consistent with pacida-
mycin nucleoside 1 (see the SI). This product was detected only
when Pac5, Pac11, and Pac13 were present. The new compound
exhibited extreme tailing under the acidic conditions employed
for LCꢀMS, possibly because of the presence of interconverting
isomers. Acetylated nucleoside 1 model compounds were shown
to be surprisingly stable toward double-bond isomerization, but
Present Addresses
Ph.D. sabbatical from Tanta University, Egypt.
†
’ ACKNOWLEDGMENT
We thank Prof. Julia Butt (UEA) and Dr. Miles Cheeseman
UEA) for useful discussions, Prof. M. J. Bibb (John Innes
(
Centre) for provision of E. coli BW25113/pIJ790 and plasmid
pIJ773, and Dr. L. M. Hill (JIC) for assistance with LCꢀMS/MS.
Funding by the Egyptian Ministry of Higher Education and the
Leverhulme Trust (F/00204/AF) is acknowledged.
’ REFERENCES
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15
the free amine 1 was never tested in those studies. Interestingly,
in the three-enzyme mixture, aminonucleoside 7 was also present
alongside residual amounts of aldehydes 9 and 10 (Figure S24).
Pac13 showed the ability to convert 7 to 1 (Figure S21). Pac13
was also incubated with hydrated pacidamycins purified from a
pac13 mutant, but the enzyme was unable to mediate the
dehydration of these species (Figure S25).
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(
(
(
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(
(
In summary, we have identified the genes that are required for
formation of pacidamycin nucleoside 1. The biosynthesis of the
nucleosidic portion follows a pathway that is different from
previously characterized nucleoside antibiotic pathways. The
biogenesis of the nucleoside has been demonstrated to start
from uridine, which is converted to its aldehyde 9 by the flavin-
dependent dehydrogenase Pac11. This is in contrast to the first
step in the biosynthesis of liposidomycin nucleoside 2, where
UMP is oxidized to aldehyde 9 through the action of LipL, a
Bai, L. Q.; Li, J. L.; Wu, M. X.; Li, R.; Xie, Z. J.; Zhou, H. C.; Zhou, X. F.;
Tan, H. R.; Deng, Z. X. J. Biol. Chem. 2009, 284, 10627.
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(
(
10) Hausinger, R. P. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 21.
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Goodenough, P. W. Trends Biochem. Sci. 2001, 26, 740.
8
non-heme iron α-ketoglutarate-dependent enzyme. To the best
(12) Zhang, W.; Ntai, I.; Bolla, M. L.; Malcomson, S. J.; Kahne, D.;
of our knowledge, this is the first example wherein the biosynth-
esis of a nucleoside antibiotic starts from the nucleoside and not
the nucleotide. Furthermore, we have demonstrated that the
dehydration is mediated by the Cupin family enzyme Pac13 and
that the transamination is catalyzed by Pac5. Both enzymes are
relatively flexible in their substrate requirements, allowing the
biosynthesis of 1 to follow a randomized order, as shown in
Scheme 2.
Kelleher, N. L.; Walsh, C. T. J. Am. Chem. Soc. 2011, 133, 5240.
(13) Heuts, D.; Scrutton, N. S.; McIntire, W. S.; Fraaije, M. W. FEBS
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Moscow) 2006, 71, S105.
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16) Gr €u schow, S.; Rackham, E. J.; Elkins, B.; Newill, P. L. A.; Hill,
L. M.; Goss, R. J. M. ChemBioChem 2009, 10, 355.
17) Ragab, A. E.; Gr €u schow, S.; Rackham, E. J.; Goss, R. J. M. Org.
Biomol. Chem. 2010, 8, 3128.
(
1
(
The generationof the unnatural pacidamycin6 thatoccurs upon
(
feeding of aldehyde 8 and amine 7, hints at the inherent substrate
16,17
flexibility within the pathway.
While the observed metabolic
plasticity leads to problems in pinpointing the timing of the
dehydration step, it will facilitate the generation of pacidamycin
analogues with altered nucleoside portions. We are carrying out
further structural and biochemical assessment of the enzymes on
this pathway and exploring the use of these enzymes in the
generation of nucleoside analogues for antiviral therapies.
’
ASSOCIATED CONTENT
S
Supporting Information. Full experimental details, LCꢀ
b
MS chromatograms, MS/MS spectra, and protein sequence
1
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dx.doi.org/10.1021/ja206163j |J. Am. Chem. Soc. 2011, 133, 15288–15291