Probing the aglycon promiscuity of an engineered glycosyltransferase

Article abstract of DOI:10.1002/anie.200803508

(Figure Presented) A sweet library: Two variants (wild-type (WT) and a triple mutant) of glycosyltransferase (GT) OleD have been shown to catalyze glycosylation of over 70 substrates, formation of O-, S- and N-glycosidic bonds, and iterative glycosylation (see scheme). Identified substrates include nucleophiles not previously known to act in GT reactions and span numerous natural product and therapeutic drug classes.

Full text of DOI:10.1002/anie.200803508

Angewandte
Chemie
DOI: 10.1002/anie.200803508
Enzyme-Catalyzed Glycosylation
Probing the Aglycon Promiscuity of an Engineered
Glycosyltransferase**
Richard W. Gantt, Randal D. Goff, Gavin J. Williams, and Jon S. Thorson*
Sugars appended to pharmaceutically important natural
products influence their key pharmacological properties
and/or molecular mechanisms of action.[1] However, studies
designed to systematically understand or exploit the role of
carbohydrates in drug discovery are often limited by the
availability of practical synthetic and biosynthetic tools.[2]
Among the contemporary options to address this limita-
tion,[3–4] chemoenzymatic glycorandomization utilizes a set of
flexible enzymes consisting of an anomeric kinase, sugar-1-
phosphate nucleotidyltransferase, and natural product glyco-
syltransferase (GT).[4–6] While chemoenzymatic glycorandom-
ization has been successfully applied to alter the natural sugar
moieties of numerous natural products,[4–8] the process
remains primarily restricted by enzyme specificity and avail-
ability of suitable GTs for the target of interest. Thus,
although there is precedent for improving non-glycosylated
therapeutics by glycoconjugation, including colchicine,[9]
mitomycin,[10] podophyllotoxin,[11] rapamycin,[12] isophosphor-
amide mustards,[13] or taxol,[14] such targets remain beyond
chemoenzymatic strategies. Recent studies on OleD, the
oleandomycin (1) GT from Streptomyces antibioticus
(Scheme 1a), revealed an enhanced triple mutant (A242V/
S132F/P67T, referred to herein as ASP) that displayed
marked improvement in proficiency and substrate promiscu-
ity.[4] To probe the synthetic utility of this enhanced catalyst
and expand upon previous reports of acceptor promiscuity for
wild-type (WT) OleD,[15] we report a comparison of the
aglycon specificities of the WT and ASP OleD variants
toward 137 drug-like acceptors. This study highlights the
ability of OleD variants to glucosylate a total of 71 diverse
acceptors and to catalyze iterative glycosylation with numer-
ous substrates, and it establishes OleD as the first multifunc-
tional GT capable of generating O-, S- and N-glycosides.
Enzymes for the study were overproduced as N-terminal
His-tag-fusions in E. coli and purified to homogeneity as
previously described.[4,5] Each member of the acceptor library
(3–139) was first assessed as a substrate for enzyme-catalyzed
Scheme 1. a) Natural reaction catalyzed by WT OleD. b) General
reaction for probing aglycon promiscuity in vitro against a panel of
137 library members; X=OH, SH, NH2, or NHR.
glucosylation with UDP-glucose (UDP-Glc) as the donor and
either WT or ASP OleD as catalyst (Scheme 1b). The library
included molecules with diverse nucleophiles and represen-
tative alkaloid, b-lactam, enediyne, non-ribosomal peptide,
polyketide, and steroid natural products. Each member was
assayed using a single “universal” assay condition (50 mm Tris
HCl (pH 8.0), 5 mm MgCl2, 0.5 mgmLÀ1 purified enzyme,
2.5 mm UDP-Glc, 1 mm aglycon, 258C, 16 h). Glycoside
production was determined by HPLC and LC-MS, and
control reactions lacking either enzyme or UDP-glucose
confirmed that products were dependent upon both enzyme
and donor.
From this first-pass analysis, enzyme-catalyzed glucosyla-
tion of 71 of the 137 library members (52%) was observed
(Figure 1). ASP provided higher conversion with 56 of the 71
substrates and, in 10 cases (14, 18, 47, 53, 66, 67, 70–73),
product was observed only with ASP. In contrast, only
polyene 57 was a unique substrate of WT OleD. Notably, of
the 71 new substrates, 4 (6, 20, 22, 42) and 21 (13, 19, 23, 26, 29,
33, 34, 37, 43–45, 47, 48, 50, 52, 53, 55, 56, 61, 63, 72) library
members exclusively contained either S- or N-based nucleo-
philes, respectively. While the first-pass LC-MS analysis could
not distinguish regio- or stereoselectivity, it is important to
note that among the subgroup of library members containing
multiple nucleophiles (34 members), 20 led to a single,
chromatographically distinct, monoglucosylated product. Per-
haps most surprising was that 13 substrates (3, 4, 6, 9, 18–21,
23, 26, 29, 33, 35) led to products with masses corresponding
to the addition of multiple glucose moieties, and within this
group, 10 (3, 6, 19–21, 23, 26, 29, 33, 35) contained only a
single heteroatom, implicating disaccharide formation
[*] R. W. Gantt, R. D. Goff, G. J. Williams, Prof. J. S. Thorson
UW National Cooperative Drug Discovery Group
Laboratory for Biosynthetic Chemistry, School of Pharmacy
University ofWisconsin-Madison
777 Highland Avenue, Madison, WI 53705 (USA)
Fax: (+1)608-262-5345
E-mail: jsthorson@pharmacy.wisc.edu
[**] The authors thank the University ofWisconsin-Madison School of
Pharmacy Analytical Facility for analytical support. This research
was supported in part by National Institutes ofHealth Grants
AI52218 and U19 CA113297.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200803508.
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Figure 1. a) Percent conversion ofeach library member with both WT and ASP OleD. Members are listed in descending order ofASP conversion
with numbering corresponding to the structures listed in (b). b) Structures ofthe corresponding library members. Compounds leading to trace
products (58–73, >5% conversion) or no conversion (74–139) are listed in the Supporting Information.
through iterative glycosylation. To confirm O-, S- and N-
glucoside formation and iterative glycosylation, and deter-
mine anomeric stereoselectivity, a select set of OleD-cata-
lyzed reactions with simple aromatic model acceptors phenol
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(8), thiophenol (6), and aniline (34) were studied in depth.
NMR characterization of products isolated from large-scale
reactions was consistent with the b-O-, S- and N-glucosides
(140–143, Scheme 2, J = 6.7, 7.8, and 9.6 Hz for H1, respec-
tively). Iterative glycosylation of model acceptor 6 was also
OleD-catalyzed iterative glycosylation. Cumulatively, the
scaffold, nucleophile, and iterative adaptability of OleD
clearly sets the stage for further engineering of custom GT
catalysts for many applications.[4,5,27]
In the context of GT promiscuity, the role of OleD in
macrolide self-resistance (1, 2, Scheme 1a) may require this
enzyme to uniquely adapt to accommodate alternative
acceptors. Yet, even with library members containing multiple
nucleophiles, the majority of reactions studied were remark-
ably regio- and stereoselective. Although other reports have
highlighted moderate promiscuity of natural product
GTs,[6,10,11,28–30] the underlying structural basis for maintaining
specificity with variant acceptors is not well understood.
Studies designed to understand the promiscuity of other
enzyme classes implicate drastic enzyme conformational
changes,[31] voluminous active sites,[32] or similar conforma-
tional binding of unrelated compounds within the active
site[33] as potential contributors to promiscuity. Consistent
with this, substrate modelling of the promiscuous GT VinC
suggested that gross molecular size and hydrogen-bonding
interactions play significant roles.[29] Future structural studies
of enhanced OleD variants bound to diverse substrates are
critical to extend our currently limited understanding of GT
specificity and catalysis. However, given the high conserva-
tion of the GT-B structural fold among natural product
GTs,[34] including OleD,[35] other examples of drastically
promiscuous GTs are likely to emerge.
Scheme 2. Products isolated from large scale enzymatic reactions of
ASP OleD with a) phenol (8), b) thiophenol (6), and c) aniline (34).
determined to be both regio- and stereoselective to provide
the 2’-O-(b-d-glucosyl)-glucoside 143 (J = 7.8 and 9.8 Hz for
H1 and H1’, respectively). Interestingly, while the kinetic
parameters for all three model substrates fell within the range
of previously reported values (Table 1 in the Supporting
Information),[4,5] the ranked order of WT OleD catalytic
efficiency (kcat/Km; thiophenol > phenol ꢀ aniline) differed
from that for ASP (phenol > aniline > thiophenol). Consis-
tent with the enhanced proficiency of ASP,[4] this mutant was
improved 25-, 5-, and 4-fold, respectively, toward phenol (8),
aniline (34), and thiophenol (6).
Received: July 18, 2008
Published online: October 16, 2008
Keywords: carbohydrates · enzymes · glycosides · glycosylation ·
.
natural products
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