C O M M U N I C A T I O N S
cloned the pubC gene from Shewanella species MR-4 and MR-7
into pET151 and overexpressed it in E. coli BL21star(DE3). The
resulting soluble His6-PubC fusion proteins (which share 97%
sequence identity) were purified from cell free extracts by Ni-NTA
chromatography. Gel filtration analysis of the purified proteins
indicated that they were dimers. We also synthesized HSP 4 in
eight steps from 1-bromo-4-chloro-butane and O-benzyl-hydoxy-
lamine using an analogous procedure to that recently reported for
HSC.6
cadaverine (1,5-diaminopentane) instead of putrescine 2.6 The linear
trimer of HSC desferrioxamine G1 was shown to be an intermediate
in this reaction and mass spectrometric evidence for the transient
accumulation of the linear dimer of HSC in the reaction was also
obtained, suggesting that this may also be an intermediate in
desferrioxamine E assembly.6 The results reported here show that
the dimeric macrocycle putrebactin 1 is assembled by ATP- and
Mg2+-dependent dimerization and subsequent macrocylization
reactions of HSP 4 in Shewanella species. In analogy to the
formation of desferrioxamine E from desferrioxamine G1, putre-
bactin 1 is formed from the linear dimer 5 of HSP, via adenylation
of the carboxyl group followed by intramolecular attack of the
amino group on the carbonyl carbon of the resulting acyl adenylate.
These reactions are catalyzed by PubC, an enzyme with nearly 50%
sequence identity to DesD. The specific formation of a macrocyclic
trimer and a macrocyclic dimer, respectively, from closely related
substrates by two highly homologous enzymes is mechanistically
intriguing. Detailed structural and kinetic studies of PubC and DesD
will be required to reveal the molecular basis for control of substrate
oligomerization by these enzymes.
Each of the two His6-PubC proteins was incubated with 4, ATP,
and Mg2+ for 5 min at 37 °C, and the reactions were stopped by
precipitation of the enzyme with tricholoracetic acid. Positive ion
LC-MS analyses of the reaction mixtures showed that two new
compounds, absent in control reactions using enzymes inactivated
by boiling, were formed, with m/z ) 391.2 and 373.1, corresponding
to the protonated ions of 5 and 1, respectively (Figure 1). The
compounds were purified from large-scale incubations of 4 with
ATP, Mg2+, and His6-PubC from Shewanella sp. MR-4 by
semipreparative reverse-phase HPLC. The molecular formulas of
the ions generated from these compounds by ESI-TOF-MS were
confirmed as C16H29N4O6+ (calculated, 373.2082; found, 373.2084)
The pubC gene resides within a conserved five-gene cluster in
the genomes of Shewanella species that is postulated to direct the
biosynthesis of 1 from putrescine, molecular oxygen, and succinyl-
CoA, the uptake of the ferric complex of 1, and the reductive
removal of iron from ferric-putrebactin. PubA, PubB, and PubC
are 46%, 40%, and 49% identical to the proteins encoded by alcA,
alcB, and alcC, respectively. These genes are involved in the
biosynthesis of alcaligin, a dihydroxylated derivative of putrebactin
that is elaborated from putrescine in Bordetella species.4 Also, PutA
shows 40% sequence identity to FauA, the ferric-alcaligin outer
membrane receptor of Bordetella species.10 Thus, it seems likely
that the biosynthesis of putrebactin and alcaligin, and the uptake
of their ferric complexes may share many similiarities in Shewanella
and Bordetella species.
+
and C16H31N4O7 (calculated, 391.2187; found, 391.2189), con-
sistent with the assignment of [1 + H]+ and [5 + H]+, respectively,
as the structures of these ions. The structure of 1 was unambiguously
confirmed by 1- and 2-D NMR analyses. ESI-MS/MS analyses of
1 and the compound with m/z ) 391.2 showed that they have
closely related structures. Both produce a daughter ion with m/z )
186.9. For both compounds, this ion fragments to give ions with
m/z ) 153.9, 140.0, 123.1, 112.1. Thus, we assign structure 5 to
the compound with m/z ) 391.2.
To further investigate the mechanism of the PubC-catalyzed
conversion of 4 to 1, we used coupled assays for AMP/ADP and
phosphate/pyrophosphate formation. These assays showed that ATP
is converted to AMP and pyrophosphate during the reaction,
strongly suggesting that PubC catalyzes reaction of 4 with ATP to
form the corresponding acyl-adenylate and pyrophosphate. Nu-
cleophilic attack of the amino group in a second molecule of 4 on
this acyl adenylate would yield 5 and AMP. Analysis of the change
in concentration of 5 and 1 with time in the PubC-catalyzed
reactions of 4, showed that 5 accumulates only transiently in the
reaction, suggesting that it is a free intermediate in the formation
of 1. To test this hypothesis, we incubated 5 (partially purified from
a large scale incubation of 4 with PubC, as described above) with
ATP, Mg2+, and His6-PubC for 2 h and 40 min at 37 °C. LC-MS
analysis of the reaction mixture, after addition of ferric iron to stop
the reaction by precipitating the enzyme and to convert 5 and 1 to
their ferric complexes, showed that 5 had been converted to 1. In
contrast, no conversion of 5 to 1 was observed in control reactions
lacking ATP or with His6-PubC inactivated by boiling prior to
addition to the reaction mixture. These data provide strong support
for the hypothesis that 5 is a free intermediate in the assembly of
1 from 4 and suggest that PubC catalyzes the reaction of ATP with
the carboxyl group of 5 to form pyrophosphate and the correspond-
ing acyl adenylate, which undergoes macrocyclization via intramo-
lecular nucleophilic attack of the ω-amino group on carboxyl group
of the adenylate, resulting in release of AMP and the formation of
1.
Acknowledgment. This work was supported by a grant from
the UK BBSRC (Grant ref. BBS/B/14450). We thank Prof. J. M.
Tiedje for supplying samples of genomic DNA from Shewanella
species MR-4 and MR-7. This paper is dedicated to the memory
of Dr. Joe Spencer.
Supporting Information Available: Experimental procedures, SDS-
PAGE analysis of His6-PubC expression and purification, spectroscopic
and enzyme assay data. This material is available free of charge via
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We recently reported that DesD catalyzes the ATP- and Mg2+
-
dependent assembly of the trimeric macrocycle desferrioxamine E
from three molecules of HSC, a homologue of HSP 4 derived from
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