Mannose-Binding Geometry
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using singly 13C-labeled Man-OMe to evaluate the geometry
of the primary Man binding of PRM-A.
[2-13C]- and [4-13C]Man-OMe gave asymmetric but distinct
intermolecular cross-peaks (Figure 2B, D), and attenuated
ones were detected in the complex with [5-13C]Man-OMe
(Figure 2E). The relative intensities and symmetry of these
intermolecular cross-peaks suggest that the C3 of Man-OMe
is most proximal to the d-alanine moiety of PRM-A, fol-
lowed by the C2, C4, and C5. In contrast, the C1 and C6 sig-
nals of Man-OMe generated only negligible or no cross-
peaks with any of the d-alanine signals of PRM-A even at
the mixing time of 500 ms (Figure 2A, F), indicating that
these positions of Man-OMe are apparently distant from the
d-alanine moiety of PRM-A. Regarding the benzo[a]naph-
thacenequinone moiety, weak but mixing time-dependent
cross-peaks were detected between the C2 signal of Man-
OMe and the C14 and C15 signals of PRM-A (for assign-
ment of these carbon signals, see Supporting Information).
The C14 signal also gave a weak cross-peaks with the C3
signal of Man-OMe. On the other hand, intermolecular
cross-peaks were hardly observed between the C1 and C4–
C6 signals of Man-OMe and carbon signals for the aromatic
region of PRM-A. Although full detection of the close con-
tacts between carbon atoms of Man-OMe and the ABC
rings of PRM-A could not be achieved due to the low 13C-
enrichment of the benzo[a]naphthacenequinone moiety, it is
quite likely that the C2 position of Man-OMe is in the clos-
est contact with the ABC rings of PRM-A.
The results of the DARR experiments collectively indi-
cate that the C2–4 region of Man is located close to the pri-
mary Man binding site in PRM-A, with the C2 and C3
carbon atoms oriented toward the ABC rings and d-alanine
moiety, respectively (Figure 3A). On the other hand, the C1
and C6 carbon atoms face outward from the binding site,
suggesting the unimportance of the acetal segment and the
6-hydroxyl group of Man in binding to PRM-A. Based on
these findings, we performed molecular modeling to esti-
mate possible interactions between PRM-A and Man from a
geometrical point of view (Figure 3B, for details of the mod-
eling, see Supporting Information). In our model, the 2-, 3-,
and 4-hydroxyl groups of Man are involved in hydrogen-
bonding interactions with PRM-A. In addition, the H2
proton of Man lies over the A ring of PRM-A at a distance
of 3.03 ꢁ, implying that the CH/p interaction between them
could also contribute to stabilization of the complex as ob-
served in many carbohydrate–receptor systems.[19] Regarding
the interaction with Ca2+ ions, our previous study suggested
the possibility that PRM-A binds Man in a Ca2+-mediated
manner through its carboxylate group.[14] Unfortunately, lack
of data regarding the geometry of Ca2+ coordination pre-
vented us from conducting further calculations in the pres-
ence of Ca2+ ions to estimate the true mode of binding.
However, it can be hypothesized that Man coordinates Ca2+
ions through the 4-hydroxyl group, which is in the proximity
of the carboxylate group of PRM-A in our binding model.
We have previously shown that the d-alanine moiety of
PRM-A was effectively 13C-enriched (ca. 70 atom% 13C) by
simultaneous addition of d-[13C3]alanine and d-cycloserine,
an inhibitor of alanine racemase, to the culture broth of
PRM-A-producing Actinomadura sp. TP-A0019.[15] Howev-
er, probably due to growth inhibition of the actinomycete by
d-cycloserine, the isolation yield of 13C-enriched PRM-A
(11.2 mg per 100 mL of culture broth) was about half that of
non-labeled PRM-A obtained by control fermentation with-
out d-cycloserine (21.5 mg per 100 mL). Thus, as a prerequi-
site for efficient and systematic DARR experiments, we
modified the feeding experiment to prepare 13C-enriched
PRM-A in sufficient quantities. d-Cycloserine inhibits the
enzymatic production of endogenous d-alanine, resulting in
effective incorporation of exogenous d-[13C3]alanine into the
d-alanine moiety of PRM-A. We assumed that suppression
of the d-alanine production could also be realized by addi-
tion of l-[13C3]alanine, which competes with endogenous l-
alanine as a substrate of alanine racemase. Moreover, this
competition could produce d-[13C3]alanine in actinomycetes,
leading to improvement of the isolation yield of 13C-en-
riched PRM-A. On the basis of these considerations, we
conducted feeding experiments using dl-[13C3]alanine with-
out d-cycloserine. To our delight, the isolation yield of 13C-
enriched PRM-A (38.8 mg per 100 mL) was markedly
higher than that in the previous feeding experiment with d-
cycloserine (11.2 mg per 100 mL), and 13C-enrichment of the
d-alanine moiety of PRM-A was almost identical for both
experiments (ca. 70 atom% 13C). Moreover, dl-[13C3]alanine
feeding was also found to increase 13C-population of the A–
E rings of PRM-A (ca. 15 atom% 13C) probably due to the
metabolic conversion of l-[13C3]alanine to [13C2]acetyl-CoA,
which constitutes the benzo[a]naphthacenequinone moiety
(Figure 1B).[18] This additional 13C enrichment, although low,
was expected to provide some information regarding intera-
tomic distances between Man and ABC rings of PRM-A, al-
lowing us to investigate the geometry of the primary Man
binding of PRM-A by using only single 13C-enriched PRM-
A. Thus, we performed 2D-DARR experiments using the
[PRM-A2/Ca2+/Man-OMe2] complexes of this 13C-enriched
PRM-A with singly 13C-labeled Man-OMe.
Although the C2, C3, and C5 signals of Man-OMe over-
lapped with the C6 signal of PRM-A in 1D-13C NMR spec-
tra, these carbon signals were clearly differentiated from
each other on the basis of the intramolecular cross-peaks be-
tween the C5 and C6 signals of PRM-A in the zoomed 2D-
DARR spectra (Supporting Information). As shown in
Figure 2, the spectra of the complexes with [2-13C]-, [3-13C]-,
[4-13C]-, and [5-13C]Man-OMe at the mixing time of 500 ms
showed significant intermolecular cross-peaks, which were
not detected in the corresponding spectra at the mixing time
of 20 ms (Supporting Information). Strong and symmetric
cross-peaks were observed between three carbon signals for
the d-alanine moiety (C17, 17-Me, C18) of PRM-A and the
C3 signal of Man-OMe (Figure 2C). The complexes with
Validation of Man binding geometry of PRM-A: The carbo-
hydrate-binding specificity of PRMs has been thoroughly ex-
amined by using BMY-28864 (1, Figure 1A), a water-soluble
Chem. Eur. J. 2013, 19, 10516 – 10525
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