Insights into the mechanism of the antibiotic-synthesizing enzyme MoeO5 from crystal structures of different complexes

Article abstract of DOI:10.1002/anie.201108002

Barrel-shaped: The enzyme MoeO5 catalyzes the transfer of the C ₁₅ moiety of farnesyl pyrophosphate to the 2-hydroxy group of 3-phosphoglycerate to give 2-(Z,E)-farnesyl-3-phosphoglycerate (FPG; ligand in the center of the shown structure). X-ray crystallographic structures showed that MoeO5 forms a triose-phosphate-isomerase barrel structure and binds FPG in a curved pocket, mainly as a result of its long λ3 loop (magenta in picture). Copyright

Full text of DOI:10.1002/anie.201108002

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Chemie
DOI: 10.1002/anie.201108002
Enzyme Mechanisms
Insights into the Mechanism of the Antibiotic-Synthesizing Enzyme
MoeO5 from Crystal Structures of Different Complexes**
Feifei Ren, Tzu-Ping Ko, Xinxin Feng, Chun-Hsiang Huang, Hsiu-Chien Chan, Yumei Hu,
Ke Wang, Yanhe Ma, Po-Huang Liang, Andrew H.-J. Wang, Eric Oldfield,* and Rey-Ting Guo*
The phosphoglycolipid antibiotic moenomycin directly blocks
bacterial cell-wall biosynthesis by inhibiting peptidoglycan
glycosyltransferases.[1] The enzyme MoeO5, encoded by the
moe gene cluster 1 in Streptomyces ghanaensis, catalyzes the
initial step of moenomycin production, in which the C15-
hydrocarbon moiety of farnesyl pyrophosphate (FPP) is
transferred to the 2-hydroxy group of 3-phosphoglycerate
(3PG), forming an ether bond (Scheme 1).[2] The reaction is
similar to that catalyzed by geranylgeranylglyceryl phosphate
synthase (GGGPS) for synthesizing archaea-type phospho-
lipids.[3] However, in contrast to the enzyme GGGPS, which
gives products with retained all-trans configuration of the
isoprenyl chain, the enzyme MoeO5 leads to a trans-to-cis
=
isomerization at the C2 C3 double bond of the transferred
farnesyl group.[4] The crystal structures of the proteins
GGGPS and the bacterial homologue PcrB reveal a triose-
phosphate isomerase (TIM)-barrel fold, which had not been
observed previously in prenyltransferases.[3,5] The sequences
of GGGPS and PcrB share 35% amino acid identity, but
MoeO5 shares only 10% identity with both enzymes (Fig-
ure S1 in the Supporting Information). Herein we report the
X-ray crystallographic structures of MoeO5 bound to the
product 2-(Z,E)-farnesyl-3-phosphoglycerate (FPG), to the
substrate analogue farnesyl thiopyrophosphate (FsPP), and to
magnesium (Mg2+) and pyrophosphate (PPi); together with
additional biochemical and bioinformatics results, these
structures shed light on the possible mechanisms of action
of this unusual enzyme.
Molecular-replacement approaches to determine the
structure of MoeO5 by using GGGPS and PcrB as search
models were not successful, thereby reflecting perhaps the
significant variations in the protein sequences. Because
MoeO5 contains no Cys residue, to solve the structure by
using multiple isomorphous replacement (MIR), we pro-
duced the mutant H97C for efficient preparation of mercury-
based MIR derivatives (Table S1 in the Supporting Informa-
tion). The other structures were solved by molecular replace-
ment (Figure S2 in the Supporting Information and Table 1;
see the Supporting Information for details). MoeO5 crystal-
lizes as a homodimer (Figure S3 and Table S2 in the
Supporting Information). The dimer interface buries
1200 ꢀ2, which is more than 10% surface area, on each
monomer and mainly involves hydrophobic residues in
helices a4 and a5. The cis-peptide of Phe140–Pro141 binds
to a Mg2+ at the molecular dyad (Figure S3 in the Supporting
Information). These helices also mediate dimerization in
GGGPS and PcrB, but in MoeO5 one of the TIM barrels is
rotated by 1808 (Figure S4 in the Supporting Information). A
more detailed description of the protein structure as well as of
the bound ligands can be found in the Supporting Informa-
tion. Despite its Ca root-mean-square deviation (r.m.s.d.) of
about 2.0 ꢀ from the GGGPS and PcrB monomers (Figure S5
in the Supporting Information), the similar protein fold with
a connecting loop (denoted l3) between strands b3 and b4
clearly places MoeO5 among this new class of TIM-barrel
prenyltransferases.
Scheme 1. The first step in moenomycin biosynthesis. The reaction is
catalyzed by MoeO5, which transfers the C15 farnesyl group from FPP
to 3PG. The carbon atoms are numbered 1–15 in FPP and 1’–3’ in
3PG. Importantly, the original all-trans configuration is converted to
2-cis,6-trans (Z,E) upon the farnesyl transfer, thereby forming 2-(Z,E)-
farnesyl-3-phosphoglycerate (FPG) and pyrophosphate ion (PPi).
[*] F. Ren,[+] Dr. C.-H. Huang, Dr. H.-C. Chan, Y. Hu, Prof. Dr. Y. Ma,
Prof. Dr. R.-T. Guo
Industrial Enzymes National Engineering Laboratory
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences, Tianjin 300308 (China)
E-mail: guo_rt@tib.cas.cn
Dr. T.-P. Ko,[+] Prof. Dr. P.-H. Liang, Prof. Dr. A. H.-J. Wang
Institute of Biological Chemistry, Academia Sinica
Taipei 11529 (Taiwan)
X. Feng, Dr. K. Wang, Prof. Dr. E. Oldfield
Department of Chemistry, University of Illinois
Urbana, IL 61801 (USA)
E-mail: eo@chad.scs.uiuc.edu
[+] These authors contributed equally to this work.
[**] This work was supported by the National Basic Research Program of
China (grant 2011CB710800 to R.T.G.), Tianjin Municipal Science
and Technology Commission (10ZCKFSY06000 to R.T.G.), and the
National Institutes of Health (AI074233 to E.O.). We thank the
National Synchrotron Radiation Research Center of Taiwan for
beam-time allocation and data-collection assistance.
The wild-type MoeO5 cocrystallized with a bound product
FPG, in which the C15 tail moiety makes a U-turn mainly at
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201108002.
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=
Table 1: Data collection and refinement statistics for the crystals of the native protein MoeO5 and of
MoeO5 in the presence of the given reagents. All positive reflections were used in the refinement. Values
in parentheses are for the outermost resolution shells.
the C8 C9 bond (Figure 1), rather
than extending straight into a non-
polar groove along helix a4 as
proposed for GGGPS and PcrB.[3,5]
The equivalent groove in MoeO5 is
obstructed by the l3 loop (Fig-
ure S6 in the Supporting Informa-
tion), which contains His97 and is
four residues longer than in
GGGPS and PcrB (Figure S1 in
the Supporting Information).[4]
This loop may act as a swinging
door for binding and enclosure of
the prenyl substrate in GGGPS,[3]
but the door is virtually locked up in
MoeO5, and the resulting small
volume seems barely able to accom-
modate a C10 group. However, sub-
stitution of a Tyr residue of GGGPS
and PcrB by Ala157 in strand b5
leads to formation of a “nook” for
the bent C15 group in MoeO5 (Fig-
ure S6 in the Supporting Informa-
tion). Neither the C10 geranyl pyro-
phosphate (GPP) nor the C20 ger-
Native
FsPP (3 h)
FsPP (overnight) PPi
3PG
Data collection
space group
unit-cell
a [ꢀ]
P21
P1
P1
P1
P1
59.0
46.6
46.7
46.7
46.8
b [ꢀ]
84.5
58.9
58.6
59.7
58.7
c [ꢀ]
59.6
58.8
58.9
59.1
59.2
a [8]
90.0
97.6
97.7
66.7
97.8
b [8]
112.3
108.4
112.2
71.1
112.6
g [8]
90.0
112.5
108.6
66.0
108.2
resolution [ꢀ]
25–1.39
(1.44–1.39)
105467
(10291)
5.1 (5.0)
97.5 (95.5)
24.8 (3.4)
6.9 (55.6)
25–1.57
(1.63–1.57)
70651
(6964)
4.0 (4.0)
96.8 (95.1)
42.4 (6.4)
3.3 (26.6)
25–1.8
(1.86–1.80)
46862
(4623)
4.0 (3.9)
96.6 (95.2)
37.3 (9.0)
3.2 (9.9)
25–1.66
(1.72–1.66)
59540
(5880)
4.0 (4.0)
96.1 (94.8)
37.1 (9.4)
3.1 (9.8)
25–1.66
(1.72–1.66)
59984
(5912)
4.0 (4.0)
96.2 (95.0)
39.9 (11.4)
3.5 (8.6)
unique reflections
redundancy
completeness [%]
average I/s(I)
Rmerge[a] [%]
Refinement
no. of reflections
99738
(8573)
69049
(6216)
46545
(4495)
59066
(5627)
59609
(5666)
[a]
Rwork (95% of data) 0.168 (0.222) 0.163 (0.203) 0.161 (0.179)
0.161 (0.184) 0.158 (0.179)
0.186 (0.227) 0.187 (0.220)
0.020
1.9
Rfree[a] (5% of data)
r.m.s.d. bonds [ꢀ]
r.m.s.d. angles [8]
dihedral angles
0.189 (0.250) 0.191 (0.243) 0.192 (0.209)
0.020
1.9
0.019
1.9
0.020
2.0
0.020
2.0
anylgeranyl
pyrophosphate
(GGPP) is a substrate for MoeO5,
and soaking with the thio-analogue
geranylgeranyl thiopyrophosphate
(GGsPP) has no effect on the
bound FPG. Clearly the cavity is
specific for FPP, and the bent con-
formation is likely to be important
in precisely positioning the C15
group for the trans-to-cis conver-
sion.
On the other hand, the binding
site for the 3PG head group is
equivalent to that for glycerol 1-
phosphate (G1P) in GGGPS (Fig-
ure S7 in the Supporting Informa-
tion).[3] The 3’-phosphate, sand-
wiched between loop b7–a7 and
helix a8’, is fastened by three back-
most favored [%] 96.2
95.8
3.0
1.2
95.8
3.2
1.0
96.2
2.8
1.0
96.0
2.8
1.2
allowed [%]
disallowed [%]
no. of non-H atoms
protein
2.6
1.2
3927
590
53
3927
467
53
3943
405
72
3932
432
62
3927
470
59
water
ligand
average B [ꢀ2][b]
protein
13.1
26.9
12.6
3VK5
18.6
31.6
21.2
3VKA
18.0
28.6
25.3
3VKB
13.9
26.1
17.6
3VKC
14.1
26.4
15.8
3VKD
water
ligand
PDB ID code
[a] R is the residual difference between the calculated and the observed structure factors (Rwork and
R
free =SjFoꢀFc/SFo) or between the measured intensities (Rmerge =SjIhjꢀIh j/nSIh, where j=1ꢀn, and
n is the number of measurements). Rfree is calculated for 5% randomly selected data which are set aside
when the structure is refined against the remaining 95% data. Rwork is calculated for these 95% data.
[b] B is the isotropic temperature of an atom.
bone NH groups (Figure 2a), and the 1’-carboxyl group,
together with His97, binds to a water molecule. When the
crystals were soaked with FsPP, partial substitution of FPG
occurred in three hours, and it was mostly replaced overnight.
The b-phosphate group of FsPP occupies the same position as
does the 3’-phosphate group of FPG, but the a-phosphate
Figure 1. Structure of the MoeO5 monomer. The central eight-stranded
b barrel and the surrounding a helices (numbered 0–8) in the ribbon
diagram are colored green and cyan, respectively. The unusual loop of
l3 is highlighted in magenta. The bound FPG and Mg–PPi complex
(from the crystal soaked with FsPP) are depicted as stick models
except Mg2+, which is shown as a blue sphere. A fraction of the other
monomer in the MoeO5 dimer is also shown, as a yellow surface
presentation.
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Figure 2. Interactions of MoeO5 with the bound ligands. a) In a stereo view, the FPG is depicted by a thick stick model with gray carbon atoms,
red oxygen atoms, and an orange phosphate atom, and the surrounding amino acids are shown as thin sticks with green carbon atoms, red
oxygen atoms, and blue nitrogen atoms. Hydrogen bonds between FPG and the protein are shown as dashed lines. The water molecule bound to
His97 is consistently observed in all crystals of the wild-type enzyme. b) The bound FsPP and Mg–PPi along with the active-site amino acids are
shown in a similar way as in (a) with the sulfur atom in FsPP shown in yellow. The coordinate bonds to the Mg2+ and other hydrogen bonds to
the ligands are shown as dashed lines. Note that PPi is closer to C1 than to C3 of the FsPP.
group is loosely bound (Figure 2b). Each active site also
shows a Mg–PPi complex adjacent to the FsPP, which may
represent an alternative disposition of the PPi moiety of FsPP
(Figure S8 in the Supporting Informa-
C3 atom, can be activated and associate with the farnesyl
cation, thereby transiently forming nerolidol (NOH) and
facilitating bond rotation (Scheme 2c). Furthermore, we find
tion). Here, the Mg2+ binds to Asp41,
which is highly conserved in MoeO5,
GGGPS, and PcrB (Table S3 in the
Supporting Information). A similar site
containing Mg2+ and PO4 was found
3ꢀ
with a 3PG soak, but PPi alone did not
bind to this site (details in the Supporting
Information). Like most other prenyl-
transferases, MoeO5 requires Mg2+ for
activity, and the network of interactions
with Mg2+ is expected to facilitate FPP
ionization, the first step in catalysis.
In the enzymes GGGPS and PcrB,
the prenyl carbocation is directly at-
tached to the C3’ hydroxy group of
G1P, and the trans-configuration is con-
served (Scheme 2a). However, for
MoeO5 Doud et al. recently proposed
catalytic mechanisms in which either
nerolidyl pyrophosphate (NPP) was
formed after ionization, thereby facili-
tating bond rotation and the trans-to-cis
conversion (Scheme 2b), or (Z,E)-FPP
was an intermediate.[4] The observation
Scheme 2. Proposed mechanisms for the TIM-barrel prenyltransferases. The reactions start by
ionization (bold arrow) of the prenyl pyrophosphate, which requires a bound Mg2+ ion. a) In
catalysis by GGGPS and PcrB, the 3’-OH group of G1P is deprotonated by the Glu167 sidechain
(Glu160 in PcrB) and associates directly with the carbocation, forming an ether bond. The
prenyl chain length varies from n=2 in GGGPS to n=5 in PcrB. b) In MoeO5, trans-FPP, (Z,E)-
FPP, and NPP all react to form FPG-like products, consistent with an ionization/isomerization
mechanism, but shorter and longer chain species do not react to form FPG. c) The residue
that trans-FPP and NPP react at the same
rate while cis-FPP reacts approximately
five times faster (Figure S9 in the Sup-
porting Information) suggests that all
three species may be involved. Another
possibility is that the His97-bound water
molecule, which is 3.6 ꢀ away from the His97 is essential for catalysis and may play a role in isomerization, possibly via NOH.
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[2] a) B. Ostash, A. Saghatelian, S. Walker, Chem. Biol. 2007, 14,
257 – 267; b) B. Ostash, E. H. Doud, C. Lin, I. Ostash, D. L.
Perlstein, S. Fuse, M. Wolpert, D. Kahne, Biochemistry 2009, 48,
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[3] J. Payandeh, M. Fujihashi, W. Gillon, E. F. Pai, J. Biol. Chem.
2006, 281, 6070 – 6078.
that H97C is an inactive mutant, supporting a key role for
His97, although (R,S)-trans-NOH (in the presence of 3PG,
PPi, and Mg2+) is not a substrate.
In summary, MoeO5 forms a TIM-barrel structure and
binds FPG in a curved pocket, mainly as a result of its long l3
loop. An FPP ionization site containing Asp41 and Mg2+ is
located nearby, and the results obtained herein are consistent
with formation of a (Z,E)-FPP intermediate. We also find that
His97 in the l3 loop is essential for activity. Except for using
Asp41 to bind Mg–PPi, the cis-bond formation catalyzed by
MoeO5 is distinct from those of other cis-prenyltransfer-
ases.[6] The crystal structures presented herein thus provide
a good starting point for future studies of this novel
mechanism of MoeO5 catalysis by using mutagenesis, kinetic
analysis, as well as other physicochemical measurements.
[4] E. H. Doud, D. L. Perlstein, M. Wolpert, D. E. Cane, S. Walker, J.
Am. Chem. Soc. 2011, 133, 1270 – 1273.
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J. Hendle, A. Huber, K. Hoda, P. Kearins, C. Kissinger, B.
Laubert, H. A. Lewis, J. Lin, K. Loomis, D. Lorimer, G. Louie, M.
Maletic, C. D. Marsh, I. Miller, J. Molinari, H. J. Muller-Die-
ckmann, J. M. Newman, B. W. Noland, B. Pagarigan, F. Park, T. S.
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Received: November 14, 2011
Revised: January 3, 2012
Published online: March 16, 2012
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Keywords: biosynthesis · enzyme catalysis · protein folding ·
protein structures · prenyltransferases
.
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