In vitro reconstitution of EryCIII activity for the preparation of unnatural macrolides

Article abstract of DOI:10.1021/ja053704n

EryCIII is a desosaminyltransferase that converts an inactive macrolide precursor to a biologically active antibiotic. It may have potential for the synthesis of unnatural macrolides with useful biological activities. However, it has been difficult to rec

Full text of DOI:10.1021/ja053704n

Published on Web 09/21/2005
In Vitro Reconstitution of EryCIII Activity for the Preparation of Unnatural
Macrolides
Yanqiu Yuan,‡,† Hak Suk Chung,‡ Catherine Leimkuhler,‡ Christopher T. Walsh,§
Daniel Kahne,‡,§ and Suzanne Walker*,†
Department of Microbiology and Genetics, HarVard Medical School, Boston, Massachusetts 02115, Department of
Biological Chemistry and Molecular Pharmacology, HarVard Medical School, Boston, Massachusetts 02115, and
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received June 6, 2005; E-mail: suzanne_walker@hms.harvard.edu
Scheme 1
Macrolides and anthracyclines are clinically important drugs
produced by streptomycetes. Both classes of natural products
contain aminosugar moieties that are attached by glycosyltrans-
ferases (Gtfs) at a late stage in the biosynthetic pathway and are
essential for biological activity (Scheme 1).1 These Gtfs are
potentially useful for the combinatorial biosynthesis or chemoen-
zymatic synthesis of structurally novel compounds with new
biological activities.2 For many years, however, all attempts to
reconstitute the activity of these Gtfs in vitro failed. Then, in 2004,
Liu and co-workers reported that DesVII, a TDP-D-desosaminyl
transferase involved in pikromycin biosynthesis, requires the
presence of an “auxiliary protein”, DesVIII, encoded in the same
gene cluster, to catalyze glycosyltransfer in vitro, although the role
of DesVIII was not clear.3 Walsh and co-workers subsequently
reported that AknS, a Gtf involved in the glycosylation of
aklavinone, requires the presence of the DesVIII homolog, AknT,
to effect glycosyltransfer in vitro.4 Walsh and co-workers proposed
that AknT functions as a regulatory subunit that increases the
catalytic activity of AknS by influencing substrate affinity.4 There
is precedent for this hypothesis because a well-characterized
We PCR amplified eryCII from a mutant strain of Saccha-
eukaryotic glycosyltransferase, â-1,4-galactosyltransferase, contains
ropolyspora erythraea NRRL 2338, ∆eryCI-60,9 and cloned the
a regulatory subunit that alters substrate affinity and selectivity.5
gene into a pET22b expression vector to obtain the plasmid pERY2,
It is important to understand the role of Gtf auxiliary proteins in
which was then transformed into E. coli BL21(DE3). Expressed
order to facilitate the use of aminosugar transferases in the
as a C-terminal His6 fusion protein, EryCII was partially purified
chemoenzymatic synthesis of macrolide and anthracycline ana-
via Ni-NTA affinity chromatography and then added to a reaction
logues. Therefore, we have been studying another aminosugar
mixture containing inactive EryCIII expressed as previously
transferase, EryCIII, which is involved in erythromycin biosynthesis
described.6 EryCIII treated with EryCII was found to be catalytically
(Scheme 1B).6,7 EryCIII catalyzes the transfer of D-desosamine to
active, effecting transfer of desosamine to RMEB. The untreated
3-R-mycarosylerythronolide B (RMEB), making erythromycin D
EryCIII sample showed no activity.
(EryD, 4). We previously reported that EryCIII can be expressed
We then examined whether AknT, shown to be required for AknS
in active form in E. coli provided that the GroEL/ES chaperonin
activity in the aclarubicin pathway, can activate purified, inactive
complex is coexpressed.6 However, the purified protein was unstable
EryCIII. AknT is 26% homologous to EryCII, and AknS is 48%
and lost activity within hours of isolation,6,8 and attempts to stabilize
homologous to EryCIII. To our surprise, we found that the addition
the activity failed. Because the erythromycin gene cluster encodes
of purified AknT to inactive EryCIII restores the activity of EryCIII
a DesVIII homolog, EryCII, we sought to determine whether EryCII
in vitro (Figure 2C).10 Thus, both EryCII and AknT can function
influenced the stability or activity of EryCIII. Studies described
as glycosyltransferase activator proteins for EryCIII, although we
below show that EryCII does activate EryCIII. Surprisingly,
note that larger amounts of AknT are required to achieve the same
however, we found that EryCIII remains active when EryCII is
level of activation (Figure 2C). Given the structural differences in
removed, which rules out models for EryCIII activation that involve
the substrates of EryCIII and AknS, this result rules out models
a stable EryCII:EryCIII complex during glycosyltransfer. We also
for activation that involve binding of substrates by the auxiliary
found that the noncognate auxiliary protein, AknT, stably activates
proteins.
EryCIII. Moreover, activated EryCIII is capable of utilizing an
To evaluate whether EryCII forms a stable complex with EryCIII
unnatural sugar donor, enabling the preparation of erythromycin
during the glycosyltransfer reaction, we prepared a plasmid
analogues. Thus, this work shows how to reconstitute the activity
encoding both native EryCII and C-terminal His6-tagged EryCIII.
of EryCIII in vitro for the synthesis of unnatural macrolides.
Protein expression was induced with IPTG, and EryCIII was
† Department of Microbiology and Genetics, Harvard Medical School.
purified away from EryCII by Ni-NTA affinity chromatography.
§ Department of Biological Chemistry and Molecular Pharmacology, Harvard
(We designate the coexpressed, purified enzyme as EryCIII*.) No
Medical School.
‡ Department of Chemistry and Chemical Biology, Harvard University.
EryCII was visible in a SDS-PAGE gel of the purified EryCIII*
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J. AM. CHEM. SOC. 2005, 127, 14128-14129
10.1021/ja053704n CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Two different ways to activate EryCIII. (A) Addition of either
EryCII or AknT to purified, inactive EryCIII in situ. (B) Coexpression of
EryCIII with EryCII and subsequent purification away from EryCII.
Figure 3. EryCIII*-catalyzed glycosylation of RMEB 3 with TDP-D-
mycaminose. Reaction conditions are as in Figure 2, except the sugar donor
and the reactions are quenched at the times indicated in the figure ([M +
H]+ calcd 720.45; observed 720.5). EB: Erythronolide B is present in the
starting material, and we have verified that it is not a substrate for EryCIII.
that EryCIII can be transiently recovered in active form when
expressed with GroEL/ES,6 and that including high concentrations
of glycerol (>15%), known to facilitate folding, restores partial
activity to the enzyme,13 we propose that EryCII or AknT facilitates
a conformational change of EryCIII from an inactive to an active
form.
Figure 2. TIC traces for glycosylation reactions catalyzed by different
batches of EryCIII (for detailed reaction conditions, see Supporting
Information): (A) 1.2 µM EryCIII; (B) 1.2 µM EryCIII, 0.4 µM EryCII;
(C) 1.2 µM EryCIII, 2.4 µM AknT; (D) 1.2 µM EryCIII*.
We are currently trying to understand in greater detail the
mechanism by which pre-exposure to EryCII or AknT activates
EryCIII for subsequent glycosyltransfer. In the meantime, this report
sets the stage for more detailed investigations of the substrate
selectivity of EryCIII, which could lead to new antibiotics with
activity against resistant microorganisms.
sample, and mass spectral analysis showed that it contained less
than 2% EryCII. Nevertheless, EryCIII* was not only fully active
(Figure 2D), but it retained activity for at least two months under
a range of different storage conditions.
The above experiments suggest that once EryCIII is activated
by EryCII, EryCII is no longer required. To assess the generality
of this observation, we treated inactive EryCIII with native AknT
for 3 h, and then purified EryCIII by Ni-NTA affinity chroma-
tography to remove AknT. This EryCIII sample, like EryCIII*,
proved to have stable enzymatic activity. Although we cannot rule
out the possibility that minute amounts of auxiliary protein influence
enzymatic activity, we note that relatively high concentrations of
other auxiliary proteins (0.25-3 equiv) seem to be required to effect
glycosyltransfer.3,4
Acknowledgment. This research was supported by the NIH
(A144854 to S.W., GM66174 to D.K., and GM20011 to C.T.W.).
We thank Kosan Biosciences for providing a gift of RMEB.
Supporting Information Available: Experimental procedures and
data for kinetics; MS characterization of products; synthesis and
characterization of TDP-sugars. This material is available free of charge
via the Internet http://pubs.acs.org.
Having identified conditions to obtain pure, active EryCIII, we
were able to characterize the kinetic behavior of the enzyme. The
apparent Km of EryCIII* for RMEB (at 1 mM TDP-D-desosamine)
was found to be 50 µM, and the apparent kcat was 1 min-1
(Supporting Information), which is comparable to the turnover
numbers observed for many other antibiotic Gtfs. We also
investigated the substrate selectivity of the purified sample and
found that D-mycaminose can be transferred to the RMEB aglycone
(3) after an overnight incubation to produce the corresponding
mycaminosyl erythromycin D derivative, erythromycin M (EryM,
5) (Figure 3).6 Thus, the activated enzyme is capable of transferring
an alternative sugar donor. This is the first report of EryCIII having
some substrate flexibility for alternative sugar donors.11 Because
the desosamine sugar contacts the 50S subunit of ribosome,12 the
discovery that an unnatural sugar can be transferred has implications
for the synthesis of new erythromycin derivatives with improved
properties.
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In conclusion, we have shown that EryCIII can be activated by
both EryCII and the noncognate Gtf auxiliary protein, AknT. We
can remove >98% of the auxiliary proteins without any apparent
loss of activity, which suggests that these proteins do not function
as regulatory subunits during the glycosyltransfer reaction. Given
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