Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases

Article abstract of DOI:10.1021/cb5009348

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6′-deoxyallose substituent occurs in a stepwise manner first at the 2′- and then the 3′-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3′-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2′-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4′-specific homologue, active site residues were identified that correlate with the 3′ or 4′ specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.

Full text of DOI:10.1021/cb5009348

Articles
pubs.acs.org/acschemicalbiology
Structural Basis of Substrate Specificity and Regiochemistry in the
MycF/TylF Family of Sugar O‑Methyltransferases.
Steffen M. Bernard,†,‡ David L. Akey,‡ Ashootosh Tripathi,‡ Sung Ryeol Park,‡ Jamie R. Konwerski,‡
Yojiro Anzai,§ Shengying Li,‡ Fumio Kato,§ David H. Sherman,‡,∥ and Janet L. Smith*,‡,⊥
†Chemical Biology Doctoral Program, University of Michigan, Ann Arbor, Michigan 48109, United States
‡Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States
§Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
∥Departments of Medicinal Chemistry, Chemistry, and Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan
48109, United States
⊥Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: Sugar moieties in natural products are
frequently modified by O-methylation. In the biosynthesis of
the macrolide antibiotic mycinamicin, methylation of a 6′-
deoxyallose substituent occurs in a stepwise manner first at the
2′- and then the 3′-hydroxyl groups to produce the mycinose
moiety in the final product. The timing and placement of the
O-methylations impact final stage C−H functionalization
reactions mediated by the P450 monooxygenase MycG. The
structural basis of pathway ordering and substrate specificity is
unknown. A series of crystal structures of MycF, the 3′-O-
methyltransferase, including the free enzyme and complexes
with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial
ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single
amino acid substitution relaxed the 2′-methoxy specificity but retained regiospecificity. The engineered variant produced a new
mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the
chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the
modeled substrate complex of a 4′-specific homologue, active site residues were identified that correlate with the 3′ or 4′
specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer.
This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of
enzymes.
Some natural product sugar O-methyltransferases can function
atural products are a valuable source of pharmacologically
Nactive compounds. The large number of stereocenters in vitro on non-natural substrates,7 and aided by high-resolution
1
structures of substrate complexes, we anticipate the ability to
more accurately assess the flexibility and specificity of this
important group of enzymes.
and complex architecture make many natural products
challenging targets for chemical synthesis. Chemoenzymatic
and bioengineering methods to generate new secondary
metabolites are becoming more versatile and have shown
promise for expanding chemical diversity.2−4 While concep-
tually straightforward, effective use of these emerging
biosynthetic approaches relies on knowledge of the substrate
specificity and reaction mechanism for each enzyme. Catalysts
for methylation at defined positions on specific sugar residues
are likely to be useful chemoenzymatic tools as they are
commonly incorporated into macrolide natural products.5
Modifications, including O-, N-, and C-methylations; acetyla-
tion; oxidation; and epimerization, result in a diverse array of
sugars not observed in primary metabolism.6 Modifications
alter the ability of macrolide natural products to interact with
targets and impact their solubility and membrane permeability.
Sugars are incorporated into natural products as nucleotide
mono- or diphosphate conjugates derived from glucose or
fructose, most commonly as thymidine diphosphate (TDP)
activated forms.6 TDP-sugars are frequently modified through
epimerization, dehydration, reduction, amination, or methyl-
ation and subsequently transferred to natural product scaffolds
by glycosyltransferases.8 Once incorporated, additional mod-
ifications including O- and N-methylation or acetylation are
common. Enzymes that catalyze TDP-sugar modification, many
Received: November 27, 2013
Accepted: February 18, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 1. Sugar methylation in natural product biosynthesis. (a) Ordered sugar O-methylation in the mycinamicin pathway. MycE catalyzes the 2′-
O-methylation of the 6′-deoxyallose of mycinamicin VI 1 to generate the javose of mycinamicin III 2. MycF catalyzes the 3′-O-methylation of javose
to form the mycinose of mycinamicin IV 3. The P450 monoxygenase, MycG, catalyzes hydroxylation and epoxide ring formation to produce the final
product mycinamicin II 4. (b) Substrates for the 4′-O-methytransferase NovP, desmethyldescarbamoyl novobiocin 5, and the 3′-O-methytransferase
TylF, macrocin 6. Modified sugars are highlighted in blue or green, and sites of methylation are denoted with red asterisks.
of which have been structurally and functionally character-
ized,5,9 may be particularly valuable in pathway engineering as
they use the common TDP nucleotide to select substrates. In
contrast, sugar modifying enzymes that act after glycosyl
transfer may interact with functional groups specific to their
substrates. Moreover, the utility of downstream sugar
methyltransferases in design or engineering of new metabolic
pathways is unknown, as structural or functional information on
their interaction their substrates is limited.
members methylate at the 3′ or 4′ hydroxyl positions.21,22
Within this subfamily, the substrate- and metal-free structure of
NovP23 and the kinetic mechanism of the homologue TylF24,25
have been reported; however the structural basis of substrate
specificity and regioselectivity is unknown.20,23 Recent
structures of natural product sugar methyltransferases, includ-
ing TcaB9,26 RebM,27 CalS11,28 MycE,20 and the MycF
homologue NovP,23 reveal a diverse array of scaffolds, as
might be expected, given their highly divergent sequences.
They share the Rossmann-like fold common to class-I
methyltransferases but differ in the elaborations that form
oligomer interfaces and confer substrate specificity.5 Notably,
few of these structures include relevant substrates. Here, we
report a series of nine crystal structures with corresponding
biochemical analysis that provide insights into substrate
specificity, pathway ordering, and mechanism of MycF that is
critical for construction of the fully elaborated macrolide
antibiotic mycinamicin II 4.29,30 Furthermore, we demonstrate
the utility of a structure-guided protein engineering approach to
generate a new macrolide antibiotic, 3′-methoxy-mycinamicin
VI 7.
Mycinamicins are produced by Micromonospora griseorubida
and are a clinically promising group of macrolide antibiotics
because they exhibit greater activity against antibiotic-resistant
bacterial strains than do clinically used antibiotics.10,11 The
mycinamicin 16-membered macrolactone core has two O-
linked modified sugars. The mycinose sugar is derived from 6′-
deoxyallose. Following transfer to the macrolide core, the sugar
is modified sequentially by two magnesium- and S-adenosyl-
methionine (SAM)-dependent methyltransferases. MycE meth-
ylates the 2′-hydroxyl of 6′-deoxyallose to form javose and
MycF methylates javose at the 3′ position to generate the
mycinose sugar (Figure 1).12 MycE and MycF catalyze
essentially the same reaction but are highly selective for their
respective substrates. Specifically, MycE catalyzes methyltrans-
fer to the 2′ sugar hydroxyl group, while MycF transfers a
methyl to the 3′ hydroxyl of a sugar that already bears a 2′
methoxy group. The distantly related MycE and MycF belong
to different branches of the class-I methyltransferase family.
Each has dozens of homologues in annotated bacterial
secondary metabolite pathways that catalyze sugar methylation
in macrolide, anthracycline, and aminocoumarin antibiotics as
well as glycopeptidolipids.13−19 We previously reported crystal
structures of MycE in binary and ternary complexes with a
cosubstrate and substrate.20 All characterized MycF subfamily
RESULTS AND DISCUSSION
■
In order to understand MycF substrate specificity, we
determined crystal structures (Table 1) for nine different states
of the protein, including the free enzyme, a binary complex with
S-adenosylhomocysteine (SAH), and four ternary complexes
with SAH and the substrate (mycinamicin III, 2), the product
(mycinamicin IV, 3), an earlier pathway intermediate
(mycinamicin VI; natural MycE substrate, 1), a non-natural
substrate from the related tylosin pathway (macrocin, 6), and
three complexes of a MycF variant. Crystals of wild type MycF
diffracted with streaky patterns that were poorly reproducible;
B
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Table 1. Crystallographic Summary
MycF WT
MycF WT
Mg, SAH
MycF WT
crystal contents
Mg - disordered lid
mycinamcin III (2), Mg, SAH
Data
P212121
space group
cell lengths (Å)
angles (deg)
dmin (Å)
C2
P22121
123.9 148.6 66.9
90 120.2 90
50.1 89.9 127.7
49.9 90.9 128.6
a
2.50 (2.50−2.49)
2.40 (2.49−2.40)
15.1 (2.9)
1.65 (1.68−1.65)
12.6 (2.1)
I/σ
13.3 (2.1)
0.080 (0.540)
5.8 (5.8)
Rsym
0.114 (0.572)
6.4 (5.9)
0.073 (0.588)
3.6 (3.6)
multiplicity
completeness
no. of unique reflections
100.0 (100.0)
36246
98.4 (89.1)
22836
99.4 (99.3)
71066
Refinement
R
work/ Rfree
0.198/0.226
0.009
0.233/0.273
0.006
0.150/0.178
0.009
RMSD bonds (Å)
RMSD angles (deg)
Ramachandran (%)
allowed
1.189
0.961
1.164
99.48
0.52
100
0
99.14
0.86
outliers
average B-factors (Å2)
protein
55.0
56.7
16.7
ligands/ions
water
49.3
56.0
28.7
48.6
49.6
25.9
PDB
4XVZ
4XVY
4X7U
MycF E35Q E139A
MycF E35Q E139A
MycF E35Q E139A
crystal contents
mycinamicin IV (3), Mg, SAH
macrocin (6), Mg, SAH
mycinamcin VI (1), Mg, SAH
Data
space group
cell lengths (Å)
angles (deg)
dmin (Å)
P212121
P212121
P212121
50.1 92.4 128.5
50.2 91.6 128.4
50.3 91.9 128.3
1.45 (1.54−1.45)
19.2 (3.2)
0.055 (0.40)
99.9 (90.9)
6.7 (4.2)
1.75 (1.85−1.75)
13.6 (4.9)
1.45 (1.50−1.45)
22.0 (4.2)
I/σ
Rsym
0.085 (0.379)
99.8 (96.8)
7.3 (7.2)
0.119 (0.591)
CC1/2 (%)
multiplicity
completeness
no. of unique reflections
6.9 (6.1)
93.0 (94.9)
105396
92.4 (63.7)
99022
97.0 (99.5)
60579
Refinement
R
work/Rfree
0.159/0.181
0.007
0.163/0.200
0.006
0.180/0.209
0.010
RMSD bonds (Å)
RMSD angles (deg)
Ramachandran (%)
allowed
1.437
1.154
1.427
100
0
100
0
99.58
0.42
outliers
average B-factors (Å2)
protein
15.7
16.8
27.3
28.1
4X7X
21.2
ligands/ions
water
22.4
32.0
29.3
32.0
PDB
4X7V
4X7W
MycF/E35Q/M56A/E139A
MycF/E35Q/M56A/E139A
MycF/E35Q/M56A/E139A
crystal contents
Mg, SAH
mycinamicin III (2), Mg, SAH
mycinamcin VI (1), Mg, SAH
Data
space group
cell lengths (Å)
angles (deg)
dmin (Å)
P212121
P212121
P212121
50.4 89.9 128.7
50.4 92.5 127.8
50.3 91.8 127.8
1.40 (1.48−1.40)
12.9 (0.9)
1.44 (1.53−1.44)
11.6 (1.4)
1.59 (1.69−1.59)
12.9 (2.0)
I/σ
Rsym
0.072 (0.901)
99.8 (50.7)
6.5 (3.3)
0.077 (0.561)
99.9 (80.8)
5.8 (3.1)
0.078 (1.03)
99.9 (87.9)
6.6 (6.6)
CC1/2 (%)
multiplicity
completeness
85.5 (43.7)
99.0 (93.6)
98.5 (96.7)
C
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Table 1. continued
MycF/E35Q/M56A/E139A
Mg, SAH
MycF/E35Q/M56A/E139A
MycF/E35Q/M56A/E139A
crystal contents
mycinamicin III (2), Mg, SAH
mycinamcin VI (1), Mg, SAH
Data
no. of unique reflections
99384
107004
79006
Refinement
R
work/Rfree
0.166/0.189
0.007
0.161/0.178
0.010
0.175/0.195
0.008
RMSD bonds (Å)
RMSD angles (deg)
Ramachandran (%)
allowed
1.251
1.467
1.354
100
0
100
0
100
0
outliers
average B-factors (Å2)
protein
18.7
14.8
31.9
4X7Y
13.7
18.7
27.2
4X7Z
19.0
33.2
32.1
4X81
ligands/ions
water
PDB
a
Values in parentheses refer to the highest resolution shell.
however, screening hundreds of crystals yielded structures of
the free enzyme, the binary SAH complex, and the ternary
substrate complex. We improved crystal quality by engineering
a double-substituted variant (E35Q/E139A) that crystallized
reproducibly, had nonstreaky diffraction, and led to high-
resolution structures of several ternary complexes. The double
substitution had no impact on catalytic activity in end-point
experiments (Table 2).
orders the remainder of the lid loop (Figure 2e). In contrast to
the full assembly of the MycF active site, the NovP/SAH
complex has a partially disordered lid loop and no metal ion.23
The ordering upon SAM binding fully buries the cosubstrate
inside MycF, consistent with the reported ordered bibi
mechanism of TylF, a MycF homologue where cosubstrate
exchange is the first and last step in the catalytic cycle.24
The ordered lid loop and helical lid domain form a funnel-
shaped substrate-entry channel with Mg2+ and SAM at the
bottom. The substrate binds with the javose sugar at the narrow
bottom of the funnel (8 Å wide). The macrolactone core rests
in the tapered upper portion of the funnel, which is 10 × 20 Å
at its widest and is formed by several hydrophobic residues
from the lid domain and lid loop (Figure 2g).
Table 2. Relative Activity of MycF Variants
MycF variant
% activity with MycF substrate (mycinamicin III, 2)
wild type
D191A
100 25
5
6
5
6
D191N
Active Site Organization. The structures of MycF provide
the first view of metal binding to a MycF/TylF family member.
A metal ion is bound in the active site in all crystallized states of
the enzyme even though no metal was included during the final
purification step. The metal is presumed to be Mg2+ based on
the octahedral coordination by oxygen ligands and the Mg2+
dependence of MycF/TylF family members.12,22,24,25 The Mg2+
binds in a negatively charged pocket where there are three
monodentate Asp ligands in all structures (Figure 2f). A fourth
Asp (191) is also a Mg2+ ligand in the SAH binary complex.
Substrate hydroxyl groups or water occupy the other sites in the
octahedral coordination shell.
The 3′- and 4′-hydroxyl groups of the mycinamicin III (2)
javose sugar coordinate the Mg2+ ion, placing the 3′-hydroxyl in
position for methylation, 4.7 Å from the SAH sulfur and 3.1 Å
from the modeled SAM methyl group. Gln246 also stabilizes
the substrate through a hydrogen bond from the 4′-hydroxy to
the amide carbonyl. In the substrate complex, the javose 3′-
hydroxyl displaces Asp191 from the Mg2+ ligand field and forms
a hydrogen bond with the Asp carboxylate. Asp191 is conserved
in the TylF/MycF family, and we hypothesized that it may be
the catalytic base to deprotonate the substrate (Figure 2f).
Asparagine and alanine substitutions at Asp191 impaired the
methyltransfer reaction nearly 100 fold, indicating that the
residue plays an important role in catalysis. (Table 2). If
Asp191 indeed deprotonates the 3′-hydroxyl, then the residual
activity of D191N and D191A could be due to deprotonation
by an active site water molecule.
M56A
301 20
11
F118Y
3
L143A
183 22
260 46
194 12
33 15
L143S
L143N
L143Q
M56A/L143A
M56A/L143S
M56A/L143N
E35Q/E139A
135
68
5
5
4
66
128 52
Overall Structure. MycF has a Rossmann-like fold
common among small-molecule methyltransferases (Figure 2a
and Supporting Information Figure 1) and, as expected, a
nearly identical structure to NovP (RMSD of 0.41 Å for 203
Cα atoms, 54% sequence identity).5,23 Both in solution and in
crystals, MycF is a dimer with an N-terminal helix (residues 6−
19) at the subunit interface (Figure 2b). A metal ion is bound
in the active site in all crystal structures, and an active site lid is
formed by an N-terminal “lid loop” (residues 25−51) as well as
an α-helical “lid domain” (residues 116−144; Figure 2d).
Substrate Binding Orders the Active site Lids. The
active site of MycF becomes progressively more ordered as the
cosubstrate and substrate bind. In the free enzyme, both parts
of the active site lid are disordered (Figure 2c). SAM/SAH
binding to MycF closes the helical lid domain over the
cosubstrate, partially orders the lid loop, and forms the binding
site for the substrate mycinamicin III (2, Figure 2d), which
D
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 2. Structure of MycF. (a) MycF monomer with SAH (orange sticks) and Mg2+ (magenta sphere) in the active site. (b) MycF dimer with N-
terminal interface. (c−e) SAM binding orders the active site lids and creates a binding pocket for mycinamicin III 2. (c) Free enzyme with disordered
lid domain and lid loop and a solvent-exposed active site Mg2+ (magenta sphere). (d) MycF-SAH. The lid domain (yellow) closes over the bound
cosubstrate, and the lid loop is partially ordered. (e) MycF-SAH-substrate. Mycinamicin III (2, green sticks) binding fully orders the lid loop (dark
blue). Spheres mark the boundaries of disordered regions. MycF is specific for the javose sugar and makes no specific contacts with the macrolactone
ring or desosamine sugar. (f) Hydrophobic interactions of MycF with the substrate macrolactone core. View is orthogonal to c−e. (g) Substrate
binding. Three Asp residues coordinate Mg2+. Mg2+ coordination of the mycinamicin III 2 3′- and 4′-hydroxy groups position the substrate for
catalysis. The SAM methyl group (modeled) is 3.1 Å from the 3′-hydroxy. Asp191 is positioned to act as the catalytic base.
Comparison to Related Methyltransferases. MycF has
an active site similar to those of other metal-dependent O-
methyltransferases (OMT), including catechol OMT,31 alfalfa
caffeoyl coenzyme A 3-OMT,32 the TDP-rhamnose 3-OMT
CalS11,28 the novobiocin 4′-OMT NovP,23 and the mycina-
micin 2′-OMT MycE.20 These metal-dependent OMTs share a
conserved SAM binding site, metal binding site, and catalytic
base position (Lys in catechol OMT and caffeoyl CoA OMT,
His in MycE, and Asp in CalS11, NovP, and MycF.) In all
cases, Mg2+ coordinates adjacent substrate hydroxyl groups and
positions the target hydroxy for methylation by SAM. Despite
the common structures of their active sites, these enzymes are
from distant branches of the class-I methyltransferase family
with highly divergent sequences, quaternary structures, and
decorations on the common methyltransferase fold.5 Among
this group, MycF is closely related only to NovP. Beyond the
fold of the catalytic core, MycF has no sequence similarity and
little structural similarity to RebM, a nonmetal dependent
E
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 3. Comparison of MycE and MycF active sites. (a) Surface representation. The active site of MycE (ref 20; 3SSN, left) is at the intersection of
three subunits (yellow, green, cyan) whereas the MycF (right; cyan) substrate binds to a single subunit. The MycE and MycF active site lids direct
substrate (cyan, green) entry on opposite sides of the catalytic Mg2+ (magenta) and SAM (orange), shown in identical orientations. (b) MycE
(yellow) and MycF (cyan) active sites. The different substrate orientations result in different sugar conformations, sugar coordination of Mg2+, and
regiochemistry of methyltransfer.
natural product sugar O-methyltransferase. This is not
surprising given the structural diversity of small-molecule
methyltransferases.
ordered, makes no protein contacts, and does not appear to
contribute to substrate specificity. Thus, MycF might accept
javose substrates bearing different macrolactones, differentially
substituted macrolactones, or nonmacrolactone hydrophobic
substituents.
It is of interest to compare the mycinamicin 2′-OMT MycE20
and the 3′-OMT MycF, as they provide the first view of
ordered sugar methylation in macrolide biosynthesis. Despite
their common active site structures, MycF and MycE have
substantially different substrate positions (Figure 3a). In MycE,
the substrate (1) 2′- and 3′-hydroxyls coordinate Mg2+ with the
sugar in a chair conformation with four axial substituents and
the 2′-hydroxy proximal to the SAM methyl. In contrast, in
MycF the substrate 3′- and 4′-hydroxyl groups coordinate Mg2
with the sugar in a different chair conformation with only one
axial substituent, and the 3′-hydroxyl near the SAM methyl
(Figure 3b). Although the MycE substrate conformation
appears less favorable (four axial substituents vs. one), the
calculated free energy difference between these two substrate
poses is only 23.9 kJ/mol (5.7 kcal/mol). This can be
rationalized by the Mg2+ coordination bonds and hydrophobic
interactions of the macrolactone core, which are more extensive
with MycE than with MycF.20
To assess the flexibility of MycF to accept alternative
substrates, we examined the activity of MycF with macrocin 6,
the javose-containing intermediate in tylosin biosynthesis.
Macrocin 6 is a 16-membered ring macrolide bearing javose
and desosamine sugars with an additional mycarose sugar
(Figure 1b). In our standard assay conditions, MycF
methylated the 3′ hydroxyl of macrocin 6 to produce the
macrolide antibiotic tylosin (Supporting Information Figure 3),
confirming that MycF tolerates changes to the macrolactone
core. As seen in the SAH-macrocin 6 ternary complex, the
differences in the macrolactone structure do not alter the
position of javose in the active site (Supporting Information
Figure 2d).
Pathway Ordering. A key motivation for our study was to
understand the structural basis for the specific order of sugar
hydroxyl group methylation in the biosynthesis of mycinamicin
and tylosin12,21,24,25 in which members of the MycF subfamily
methylate the 3′-hydroxyl only after methylation of the 2′-
hydroxyl of 6′-deoxyallose (creating the javose moiety). How
does MycF distinguish the substrate mycinamicin III 2 from
mycinamicin VI 1, which is not a substrate,12 as these molecules
differ only in the 2′ substituent (methoxy vs hydroxy)? It was
Substrate Selectivity. The MycF ternary complex with
SAH and the natural substrate mycinamicin III 2 suggests that
MycF may be active with alternative substrates. The macro-
lactone is located in a hydrophobic region at the opening of the
active site funnel (Supporting Information Figure 2a) where it
is partially solvent-exposed and forms no specific contacts with
MycF (Figure 2g). The terminal desosamine sugar is less well
F
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 4. MycF activity with MycE substrate (mycinamicin VI 1). (a) MycF active site with 2′-methoxy specificity pocket. Residues selected for
substitution experiments are shown in gray sticks. (b) HPLC chromatograms of MycF variants with the MycE substrate (mycinamicin VI 1). Wild
type MycF and MycF/M56A show production of a new product that has a retention time similar to mycinamicin III 2. (c) Mass spectrum of HPLC-
purified product of MycF/M56A reaction with the MycE substrate. The M+H peak for the product has a mass comparable to that for the MycE
product (monoisotopic mass of mycinamicin III 2 681.4 Da compared to 682.4 Da for the M+H peak). (d) Structure of 3′-methoxy-mycinamicin VI
7. COSYand HMBCAD correlations are indicated; NMR spectra are in the Supporting Information.
not immediately apparent from the structures why MycF does
not methylate the MycE substrate mycinamicin VI 1.
point assay (Table 2). Several double-substituted MycF variants
lacked synergistic or additive effects in the end point assay with
the natural substrate 2, and no activity was observed with the
MycE substrate 1 in the standard assay (Table 2). Steady state
kinetic constants were similar for the wild type (Km 10.9 2.9
The javose 2′-methoxy binds in a hydrophobic pocket
created by Met56, Phe118, Leu143, and Val141 (Figure 4a).
Reasoning that this pocket might be too hydrophobic for a 2′-
hydroxyl group, we substituted alanine or a polar side chain for
each of three hydrophobic amino acids in the pocket (Met56,
Phe118, Leu143). None of the variants had detectable activity
with mycinamicin VI 1 under standard assay conditions
although, interestingly, four of the substitutions increased
activity with the natural substrate mycinamicin III 2 in our end
μM, Vmax 0.08
0.01 μMs1−, kcat 0.27 s−1) and the M56A
variant (Km 6.4 1.8 μM, Vmax 0.04 0.002 μMs1−, kcat 0.13
s−1). However, the wild type MycF had decreased initial
velocity at high substrate concentrations, suggestive of substrate
inhibition, whereas the M56A variant exhibited no substrate
inhibition and thus appeared “hyper-active” (Supporting
G
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Information Figure 4). As SAM/SAH binds beneath the
substrate (Figure 2e), we hypothesize that at high substrate
concentrations, product release is followed by new substrate
binding faster than SAM replaces SAH in the active site.
Substrate (or product) binding stabilizes the fully closed
conformation of the active site lid (Figure 2e), which may slow
both cosubstrate exchange and the rate of catalysis, explaining
the observed greater activity of M56A in the end point assay
(Table 2). Met56 is adjacent to SAM (Figure 4a), and
substitution with Ala may allow SAH/SAM exchange in the
presence of substrate or product.
Remarkably, the crystal structure of MycF in a ternary
complex with SAH and the MycE substrate shows that
mycinamicin VI 1 binds similarly to the authentic MycF
substrate mycinamicin III 2 with 3′- and 4′-hydroxy
coordination of Mg2+ (Supporting Information Figure 2c).
However, the sugar is shifted ∼1 Å deeper into the binding
pocket and rotated ∼15° toward SAM (Supporting Information
Figure 5a), positioning the polar 2′-hydroxyl group outside the
hydrophobic methoxy binding pocket. The Met56 side chain
appears to exclude the authentic substrate from this position by
a steric clash with the 2′-methoxy. Residual positive Fo−Fc
electron density indicates that the sugar samples multiple
conformations. The predominant position is incompatible with
the SAM methyl group (∼2.6 Å from the 3′-hydroxy and ∼2.7
Å from the 2′-hydroxy, Supporting Information Figure 5b,c).
Thus, mycinamicin VI 1 may be excluded from the MycF active
site when SAM is bound, or it may bind in a nonproductive
position in the presence of SAM.
Generation and Characterization of a New Mycina-
micin Compound. The binding pose of the MycE substrate in
the MycF active site suggests that MycF should methylate the
MycE substrate, despite the lack of activity under standard assay
conditions. At high concentrations, MycF converted approx-
imately 10% of mycinamicin VI 1 (MycE substrate) to a
previously unreported product (Figure 4b). Under the same
conditions, the M56A variant exhibited 100% conversion of
mycinamicin VI to the new product (Figure 4b) with a mass
relative to the starting material consistent with a single
methylation (+14 Da, Figure 4c) but a slightly different
HPLC retention time than the singly methylated mycinamicin
III product of MycE (Figure 4b). We confirmed with
multidimensional NMR that the product is 3′-methoxy-
mycinamicin VI 7, a new mycinamicin analog methylated at
the 3′-hydroxy (but not the 2′-hydroxy; Table 3, Figure 4d, and
Supporting Information). Remarkably, the engineered enzyme
had reduced substrate selectivity but retained regiospecificity;
i.e. each substrate produced only a single product. These results
demonstrate the potential utility of the MycF M56A variant in
engineered production strains. Although the variant does not
exhibit increased catalytic efficiency relative to the wild type,
the decrease in substrate inhibition results in increased product
formation over time.
Table 3. Chemical Shift Assignments for 3′-Methoxy-
mycinamicin VI
atom
δC
δH, multi (J in Hz)
COSY
HMBC
1
2
3
4
5
6
7
8
9
121.5
151.4
41.7
88
5.93, d (15.4)
3
6.54, dd (10.1, 15.4) 2, 4
2.76, m
3.36, m
1.22, m
1.72, m
2.47, m
3, 5, 18
4
34
19
8
33.18
45.3
203.1
123.7
141.2
133.5
141.2
49.3
74.1
23.6
9.7
7, 20
10
6.46, d (15.0)
11
11
7.01, dd (10.9, 15.0) 10, 12
6.24, dd (10.9, 15.4) 11, 13
12
13
6.02, dd (9.3, 15.4)
2.49, m
12
14
13, 15, 21
15
4.91, m
14, 16
16
1.59, m
15, 17
17
0.9, t (7.3, 14.6)
1.23, d (6.7)
0.99, d (6.8)
1.14, d (7.0)
3.68, m
16
18
19.4
17.6
17.7
68.4
105.7
70
4
19
6
5, 7
20
8
7, 9
21
14
C1′
C2′
C3′
C4′
C5′
C6′
N(CH3)2
C1″
C2″
C3″
C4″
C5″
C6″
C7″
4.28, d (7.3)
3.16, dd (7.3, 9.8)
2.53, m
C2′
5
C1′, C3′
C2′, C4′
C3′
66.5
29.56
69.5
21.2
40.4
101.9
72.7
82.6
73.9
70.9
17.9
61.7
1.74, m
3.55, m
1.18, d (3.2)
2.29, m
C5′
C5′, C4′
C3′, N(CH3)2
21
4.5 d, (7.8)
3.41, m
C2″
C1′, C3′
C2″, C4″
C3″, C5″
C4″
3.65, m
3.18, m
3.15, m
1.19, d (3.2)
3.58, m
C4″, C5″
C3″
complex. The hydrophobic 2′-methoxy substituent in the
authentic substrate can engage other surfaces of the hydro-
phobic pocket that are less accommodating to a 2′-hydroxy
substituent, explaining the difference in activity of MycF/M56A
on the two substrates.
Enzymatic production of 3′-methoxy-mycinamicin VI
allowed us to test for the first time whether MycE can accept
substrates with a 3′-methoxy sugar. MycE was unable to
produce the doubly methylated mycinamicin IV 3 from 3′-
methoxy-mycinamicin VI 7 (Supporting Information Figure 8).
A hydrogen bond between the mycinamicin VI 3′-hydroxy and
MycE His180 (ref 20, 3SSN) selects the 3′-hydroxy substrate.
These results demonstrate how both MycE and MycF
contribute to reaction ordering in the tailoring steps of
macrolide biosynthesis. The importance of this ordering is
underscored by the observation that the MycG monoxygenase
that completes mycinamicin biosynthesis requires both the 2′-
and 3′-methoxy groups on the mycinose sugar.33
MycF/M56A binds Mg2+ and SAH identically to the wild
type, but both substrates, mycinamicin III and mycinamicin VI
(Supporting Information Figure 6), occupy the deep position
observed in wild type MycF for the MycE substrate. The
position of the Met56 side chain is occupied by a DMSO
molecule (mycinamicin solvent) in the mycinamicin VI
complex (Supporting Information Figures 5d and 7) and by
the 2′-methoxy in the mycinamicin III complex. The position of
both substrates is incompatible with the SAM cosubstrate;
therefore both substrates must move in the substrate−SAM
Regiospecificity in MycF/TylF Family Members.
Proteins in the MycF/TylF/NovP subfamily of the class-I
methyltransferases have high sequence identity (∼50% or
greater) and are expected to have structures as similar to MycF
as is NovP (0.4 Å rmsd, 54% identity). Thus, the structures of
H
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 5. Regiochemistry of methyltransfer in MycF homologues. (a) Model of NovP substrate complex. The NovP 4′ O-MT is in dark blue,
substrate DDN 5 in gray, and SAM in orange. (b) Structure of MycF−substrate complex with MycF in cyan, mycinamicin III 2 in green, and SAM in
orange. (c) Partial sequence alignment of MycF homologues (MycF numbering). All MycF/TylF family members with known substrates are
grouped based on the product sugar, shown at right. Tryptophan at position 49 is strictly conserved among 4′-specific enzymes, as is glutamine at
position 246 in 3′-specific enzymes. The conserved tryptophan contributes to the hydrophobic pocket for the 5′-methyl group (shown in a). MycF,
mycinamicin 3′-O-methyltransferase;34 TylF, tylosin 3′-O-methyltransferase;35 ChmMII, chalcomycin 3′-O-methyltransferase;18 NovP, novobiocin
4′-O-methyltransferase; CouP, coumermycin 4′-O-methyltransferase;17 CloP, clorobiocin 4′-O-methyltransferase;14 BusH, butenyl-spinosyn 4′-O-
methyltransferase;13 SpnH, spinosyn 4′-O-methyltransferase;16 MtfC, Mycobacterium avium glycopetidolipid 4′-O-methyltransferase; MtfB,
Mycobacterium avium glycopetidolipid 4′-O-methyltransferase; Rmt4, Mycobacterium chelonae glycopetidolipid 4′-O-methyltransferase; ElmMIII,
elloramycin 4′-O-methyltransferase;19 SnogL, nogalamycin 4′-O-methyltransferase.15
subfamily members and their active sites can be modeled with a
high degree of confidence. The 13 experimentally characterized
subfamily members are sugar 3′- and 4′-OMTs that act after the
glycosyltransfer step in the biosynthesis of secondary
metabolites. We modeled the substrate complex for the 4′-
OMT NovP (2WK1),23 the only other subfamily member of
known structure. The substrate DDN (5) was manually
positioned in the NovP active site constrained by the
methylation site and Mg2+ coordination geometry of MycF
(Figure 5a). The lid loop of MycF guided positioning of the
DDN (5) aminocoumarin, as the corresponding residues are
disordered in the NovP structure. Positioning the 4′-hydroxy
for methylation and the 3′-hydroxy as a Mg2+ ligand required
that the novobiose sugar be flipped in the active site relative to
the orientation of javose in MycF (Figure 5a,b). The novobiose
sugar makes no specific contacts with amino acids apart from
the Asp191 putative catalytic base. The strict regiospecificity of
the sugar OMTs results from the relative positions of the
hydrophobic macrolactone binding site, metal site, and metal
ligands that position the substrate 3′- or 4′-hydroxy near the
SAM methyl.
With MycF (3′-OMT) and NovP (4′-OMT) as prototypes,
we grouped the aligned sequences of the 13 experimentally
characterized subfamily members based upon the substrate
structures and sites of methylation (Figure 5c). Two amino
acids correlate with the regiospecificity of methyl transfer and
interact with the substrate in the MycF structure and NovP
model. MycF Gln246 forms a hydrogen bond with the sugar 4′-
hydroxy and is conserved among the 3′-OMTs, all of which
have sugar 4′-hydroxyl substrates. The Gln246 interaction
I
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
excludes substrates with 4′-alkoxy substituents. A smaller polar
amino acid (Asp or Thr) occupies the analogous position in the
4′-specific enzymes. NovP Trp58, conserved in the 4′-specific
enzymes, forms a hydrophobic pocket for the 5′-methyl
substituent in all known substrates of the 4′-OMTs. The 3′-
specific enzymes have a conserved tyrosine or phenylalanine at
the analogous position (Tyr49 in MycF). Given the variability
of the substrate sugars, the strict correlation of these two amino
acids with the regiospecificity of the enzymes is striking and will
be useful in the identification and annotation of new natural
product biosynthetic pathways.
On the basis of the MycF structures and NovP model, we
propose guidelines to predict the substrate specificity and
regioselectivity of the several dozen MycF/TylF family
members with uncharactized activities (Figure 5). MycF is
selective for substrates with 3′ and 4′ syn-hydroxyl groups that
are on the opposite face of the sugar from the glycosidic bond,
as found in the mycinose sugar of mycinamicn or tylosin.
Although methoxy groups can serve as metal ligands, as
observed in the MycF product (mycinamicin IV, 3) complex,
MycF cannot accommodate 4′-methoxy groups in the active
site. In contrast, NovP requires anti-hydroxyl groups at the 3′
and 4′ positions like those in rhamnose and also requires that
the methylation site and glycosidic bond occur on the same face
of the sugar ring. Further, NovP and related 4′-O-
methyltransferases can accommodate 3′-methoxy substituents
as Mg2+ ligands, consistent with the 2′-,3′-,4′-hydroxyl group
methylation order in the spinosyn pathway.21
We determined a series of structures that provide insight into
the substrate specificity, mechanism, and regiochemistry of a
common subfamily of sugar O-methyltransferases. The location
of the 16-membered macrolactone ring and open active site
suggests that this subfamily of methyltransferases may accept a
variety of hydrophobic substrates bearing sugar moieties. On
the basis of the substrate complex, we propose a model of the
substrate position in 4′-specific members of this subfamily and
identify key amino acids that can be used in the annotation of
new biosynthetic pathways.
The MycF structures allowed us to identify an amino acid
substitution that decreased substrate inhibition and relaxed the
2′-methoxy specificity to produce a new mycinamicin analog,
while maintaining the regiospecificity of the enzyme. This is the
first example of the rational engineering of a natural product
sugar O-methyltransferase and demonstrates the utility of a
structure-guided approach to producing new compounds.
MycF variants with decreased substrate inhibition could be
used in the development of high-yield strains for production-
scale fermentation of macrolide antibiotics with the mycinose
sugar including mycinamicins or tylosin. The synergistic use of
structural information on glycosyltransferases and sugar-
modifying enzymes including the MycE and MycF families of
sugar O-methyltransferases will enable their use for chemo-
enzymatic synthesis of novel complex natural product
analogues.
primers in Supporting Information Table 1). MycF variants
were expressed and purified in the same manner as WT MycF.
Crystallization. Initial crystals of MycF were grown by
hanging-drop vapor diffusion with a 1:1 ratio of protein
solution (10 mg mL−1 MycF in 20 mM Tris 8.0, 150 mM NaCl
10% glycerol, 1 mM SAH) and well solution (20−30% PEG
3350, 100 mM BisTrisPropane pH 6.5). These crystals were
used to solve structures of the apo enzyme, SAH binary
complex, and SAH substrate ternary complex. Crystals of MycF
E35Q/E139A were grown using a well solution containing 20−
30% PEG 5000 MME, 100 mM ammonium acetate, and 100
mM BisTrisPropane pH 6.5. These crystals were used to solve
structures of MycF in complex with mycinamicin IV 3,
mycinamicin VI 1, and macrocin 6 and to redetermine the
two previously solved complex structures. Apart from the
double substitution, the redetermined structures were of similar
quality and identical to the wild type complex structures.
Crystals of the E35Q/M56A/E139A variant were grown under
the same conditions as the E35Q/E139A variant by micro-
seeding. For the mycinamicin III 2, mycinamicin IV 3,
mycinamicin VI 1, and macrocin 6 complexes, the crystals
were soaked 4−6 h in well solution with 10 mM ligand.
Crystals were harvested on MicroMesh mounts (MiTeGen),
cryoprotected in well solution plus 10% glycerol and flash
cooled in liquid N2.
Enzyme Assays. The end point assay was done according
to published protocol12 with a 100 μL reaction mix containing
50 mM Tris buffer (pH 8.0), 5 μM MycF, 250 μM substrate 2,
10 mM MgCl2. The reaction was initiated by the addition of
750 μM SAM, incubated 30 min at 30 °C, and quenched by the
addition of 100 μL methanol. Mycinamicin VI 1 reactions were
performed in the same manner with 20 μM MycF and
incubated 20 h. MycE reactions were performed under the
same conditions with 5 μM MycE in place of MycF.
Precipitated protein was removed by centrifugation, and the
components of the reaction mixture were separated by reverse
phase HPLC; absorbance was monitored at 280 nm. Integrated
peak areas for the substrate and product were used to calculate
percent conversion and normalized to the wild type MycF
activity. Reactions were performed in triplicate. Components of
reaction mixtures were validated with authentic standards of
mycinamicin VI 1, mycinamicin III 2, and mycinamicin IV 3.
The product of the MycF reaction with mycinamicin III 2 and
SAM has been characterized by LC-MS and reported
previously.12
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting Information methods, tables, and figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
METHODS
■
Atomic coordinates and structure factors have been deposited
in the Protein Data Bank under the following PDB accession
codes: 4XVZ (MycF), 4XVY (MycF-SAH), 4X7U (mycinami-
cin III complex), 4X7V (mycinamicin IV complex), 4X7W
(mycinamicin VI complex) 4X7X (macrocin complex), 4X7Y
(MycF/M56A-SAH), 4X7Z (MycF/M56A mycinamicin III
complex), and 4X81 (MycF/M56A mycinamicin VI complex).
Protein Expression, Purification, and Mutagenesis.
Recombinant expression of native MycF has been described
previously.12 The single substitution variants were made with
the Quik Change II Site-Directed Mutagenesis Kit (Stratagene)
and the E35Q/E139A double mutant with the QuikChange
Lightning Multi Site Directed Mutagenesis Kit (Strategene)
following the manufacturer’s protocols (forward and reverse
J
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
(14) Pojer, F., Li, S. M., and Heide, L. (2002) Molecular cloning and
sequence analysis of the clorobiocin biosynthetic gene cluster: new
insights into the biosynthesis of aminocoumarin antibiotics. Micro-
biology 148, 3901−3911.
(15) Torkkell, S., Kunnari, T., Palmu, K., Mantsala, P., Hakala, J., and
Ylihonko, K. (2001) The entire nogalamycin biosynthetic gene cluster
of Streptomyces nogalater: characterization of a 20-kb DNA region and
generation of hybrid structures. Mol. Genet. Genomics 266, 276−288.
(16) Waldron, C., Matsushima, P., Rosteck, P. R., Broughton, M. C.,
Turner, J., Madduri, K., Crawford, K. P., Merlo, D. J., and Baltz, R. H.
(2001) Cloning and analysis of the spinosad biosynthetic gene cluster
of Saccharopolyspora spinosa. Chem. Biol. 8, 487−499.
(17) Wang, Z. X., Li, S. M., and Heide, L. (2000) Identification of the
coumermycin A(1) biosynthetic gene cluster of Streptomyces rishiriensis
DSM 40489. Antimicrob. Agents Chemother. 44, 3040−3048.
(18) Ward, S. L., Hu, Z. H., Schirmer, A., Reid, R., Revill, P., Reeves,
C. D., Petrakovsky, O. V., Dong, S. D., and Katz, L. (2004)
Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis:
Novel features of an unusual ketolide produced through expression of
the chm polyketide synthase in Streptomyces fradiae. Antimicrob. Agents
Chemother. 48, 4703−4712.
(19) Patallo, E. P., Blanco, G., Fischer, C., Brana, A. F., Rohr, J.,
Mendez, C., and Salas, J. A. (2001) Deoxysugar methylation during
biosynthesis of the antitumor polyketide elloramycin by Streptomyces
olivaceus - Characterization of three methyltransferase genes. J. Biol.
Chem. 276, 18765−18774.
(20) Akey, D. L., Li, S., Konwerski, J. R., Confer, L. A., Bernard, S.
M., Anzai, Y., Kato, F., Sherman, D. H., and Smith, J. L. (2011) A new
structural form in the SAM/metal-dependent O-methyltransferase
family: MycE from the mycinamicin biosynthetic pathway. J. Mol. Biol.
413, 438−450.
(21) Kim, H. J., White-Phillip, J. A., Ogasawara, Y., Shin, N., Isiorho,
E. A., and Liu, H. W. (2010) Biosynthesis of spinosyn in
Saccharopolyspora spinosa: synthesis of permethylated rhamnose and
characterization of the functions of SpnH, SpnI, and SpnK. J. Am.
Chem. Soc. 132, 2901−2903.
(22) Freel Meyers, C. L., Oberthur, M., Xu, H., Heide, L., Kahne, D.,
and Walsh, C. T. (2004) Characterization of NovP and NovN:
completion of novobiocin biosynthesis by sequential tailoring of the
noviosyl ring. Angew. Chem., Int. Ed. Engl. 43, 67−70.
(23) Gomez Garcia, I., Stevenson, C. E., Uson, I., Freel Meyers, C. L.,
Walsh, C. T., and Lawson, D. M. (2010) The crystal structure of the
novobiocin biosynthetic enzyme NovP: the first representative
structure for the TylF O-methyltransferase superfamily. J. Mol. Biol.
395, 390−407.
AUTHOR INFORMATION
Corresponding Author
*E-mail: janetsmith@umich.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grant DK042303 to J.L.S.
and National Institutes of Health grant GM078553 and the
Hans W. Vahlteich Professorship to D.H.S. S.M.B. acknowl-
edges training grant support from the University of Michigan
Chemistry−Biology Interface (CBI) training program (NIH
grant 5T32GM008597). GM/CA has been funded in whole or
in part with federal funds from the National Cancer Institute
(Y1-CO- 1020) and the National Institute of General Medical
Sciences (Y1-GM-1104). Use of the Advanced Photon Source
was supported by the U.S. Department of Energy, Basic Energy
Sciences, Office of Science, under contract No. DE-AC02-
06CH11357.
REFERENCES
■
(1) Newman, D. J., and Cragg, G. M. (2007) Natural products as
sources of new drugs over the last 25 years. J. Nat. Prod. 70, 461−477.
(2) Park, S. R., Han, A. R., Ban, Y. H., Yoo, Y. J., Kim, E. J., and Yoon,
Y. J. (2010) Genetic engineering of macrolide biosynthesis: past
advances, current state, and future prospects. Appl. Microbiol.
Biotechnol. 85, 1227−1239.
(3) Mortison, J. D., and Sherman, D. H. (2010) Frontiers and
opportunities in chemoenzymatic synthesis. J. Org. Chem. 75, 7041−
7051.
(4) Hansen, D. A., Rath, C. M., Eisman, E. B., Narayan, A. R.,
Kittendorf, J. D., Mortison, J. D., Yoon, Y. J., and Sherman, D. H.
(2013) Biocatalytic synthesis of pikromycin, methymycin, neo-
methymycin, novamethymycin, and ketomethymycin. J. Am. Chem.
Soc. 135, 11232−11238.
(5) Liscombe, D. K., Louie, G. V., and Noel, J. P. (2012)
Architectures, mechanisms and molecular evolution of natural product
methyltransferases. Nat. Prod. Rep. 29, 1238−1250.
(6) Thibodeaux, C. J., Melancon, C. E., and Liu, H. W. (2008)
Natural product sugar biosynthesis and enzymatic glycodiversification.
Angew. Chem., Int. Ed. Engl. 47, 9814−9859.
(7) Zhang, C., Albermann, C., Fu, X., Peters, N. R., Chisholm, J. D.,
Zhang, G., Gilbert, E. J., Wang, P. G., Van Vranken, D. L., and
Thorson, J. S. (2006) RebG- and RebM-catalyzed indolocarbazole
diversification. ChemBioChem. 7, 795−804.
(8) Thibodeaux, C. J., Melancon, C. E., and Liu, H. W. (2007)
Unusual sugar biosynthesis and natural product glycodiversification.
Nature 446, 1008−1016.
(9) Singh, S., Phillips, G. N., Jr., and Thorson, J. S. (2012) The
structural biology of enzymes involved in natural product
glycosylation. Nat. Prod. Rep. 29, 1201−1237.
(10) Satoi, S., Muto, N., Hayashi, M., Fujii, T., and Otani, M. (1980)
Mycinamicins, new macrolide antibiotics 0.1. taxonomy, production,
isolation, characterization and properties. J. Antibiot. 33, 364−376.
(11) Anzai, Y., Salto, N., Tanaka, M., Kinoshita, K., Koyama, Y., and
Kato, F. (2003) Organization of the biosynthetic gene cluster for the
polyketide macrolide mycinamicin in Micromonospora griseorubida.
FEMS Microbiol. Lett. 218, 135−141.
(24) Bauer, N. J., Kreuzman, A. J., Dotzlaf, J. E., and Yeh, W. K.
(1988) Purification, characterization, and kinetic mechanism of S-
adenosyl-L-methionine: macrocin O-methyltransferase from Strepto-
myces fradiae. J. Biol. Chem. 263, 15619−15625.
(25) Kreuzman, A. J., Turner, J. R., and Yeh, W. K. (1988) Two
distinctive O-methyltransferases catalyzing penultimate and terminal
reactions of macrolide antibiotic (tylosin) biosynthesis. Substrate
specificity, enzyme inhibition, and kinetic mechanism. J. Biol. Chem.
263, 15626−15633.
(26) Bruender, N. A., and Holden, H. M. (2012) Probing the
catalytic mechanism of a C-3′-methyltransferase involved in the
biosynthesis of D-tetronitrose. Protein Sci. 21, 876−886.
(27) Singh, S., McCoy, J. G., Zhang, C., Bingman, C. A., Phillips, G.
N., Jr., and Thorson, J. S. (2008) Structure and mechanism of the
rebeccamycin sugar 4′-O-methyltransferase RebM. J. Biol. Chem. 283,
22628−22636.
(28) Singh, S., Chang, A., Helmich, K. E., Bingman, C. A., Wrobel, R.
L., Beebe, E. T., Makino, S. I., Aceti, D. J., Dyer, K., Hura, G. L.,
Sunkara, M., Morris, A. J., Phillips, G. N., Jr., and Thorson, J. S. (2013)
Structural and Functional Characterization of CalS11, a TDP-
Rhamnose 3′-O-Methyltransferase Involved in Calicheamicin Biosyn-
thesis. ACS Chem. Biol. 8, 1632−1639.
(12) Li, S., Anzai, Y., Kinoshita, K., Kato, F., and Sherman, D. H.
(2009) Functional analysis of MycE and MycF, two O-methyl-
transferases involved in the biosynthesis of mycinamicin macrolide
antibiotics. ChemBioChem. 10, 1297−1301.
(13) Hahn, D. R., Gustafson, G., Waldron, C., Bullard, B., Jackson, J.
D., and Mitchell, J. (2006) Butenyl-spinosyns, a natural example of
genetic engineering of antibiotic biosynthetic genes. J. Ind. Microbiol.
Biotechnol. 33, 94−104.
(29) Anzai, Y., Li, S., Chaulagain, M. R., Kinoshita, K., Kato, F.,
Montgomery, J., and Sherman, D. H. (2008) Functional analysis of
K
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis
of mycinamicin macrolide antibiotics. Chem. Biol. 15, 950−959.
(30) Li, S., Tietz, D. R., Rutaganira, F. U., Kells, P. M., Anzai, Y.,
Kato, F., Pochapsky, T. C., Sherman, D. H., and Podust, L. M. (2012)
Substrate recognition by the multifunctional cytochrome P450 MycG
in mycinamicin hydroxylation and epoxidation reactions. J. Biol. Chem.
287, 37880−37890.
(31) Vidgren, J., Svensson, L. A., and Liljas, A. (1994) Crystal
structure of catechol O-methyltransferase. Nature 368, 354−358.
(32) Ferrer, J. L., Zubieta, C., Dixon, R. A., and Noel, J. P. (2005)
Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransfer-
ase. Plant Physiol. 137, 1009−1017.
(33) Anzai, Y., Tsukada, S., Sakai, A., Masuda, R., Harada, C.,
Domeki, A., Li, S., Kinoshita, K., Sherman, D. H., and Kato, F. (2012)
Function of cytochrome P450 enzymes MycCI and MycG in
Micromonospora griseorubida, a producer of the macrolide antibiotic
mycinamicin. Antimicrob. Agents Chemother. 56, 3648−3656.
(34) Inouye, M., Suzuki, H., Takada, Y., Muto, N., Horinouchi, S.,
and Beppu, T. (1994) A gene encoding mycinamicin III O-
methyltransferase from Micromonospora griseorubida. Gene 141, 121−
124.
(35) Cundliffe, E., Bate, N., Butler, A., Fish, S., Gandecha, A., and
Merson-Davies, L. (2001) The tylosin-biosynthetic genes of
Streptomyces fradiae. Antonie Van Leeuwenhoek 79, 229−234.
L
DOI: 10.1021/cb5009348
ACS Chem. Biol. XXXX, XXX, XXX−XXX