Characterization of LipL as a non-heme, Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that generates uridine-5′-aldehyde during A-90289 biosynthesis

Article abstract of DOI:10.1074/jbc.M110.203562

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG: UMP dioxygenase that produces uridine-5′-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O₂, stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K_m^α-KG = 7.5 μM, K_m^UMP = 14 μM, and k_cat ≈ 80 min ^-1. The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Full text of DOI:10.1074/jbc.M110.203562

Enzymology:
Characterization of LipL as a Non-heme,
Fe(II)-dependent α-Ketoglutarate:UMP
Dioxygenase That Generates Uridine-5 ′
-aldehyde during A-90289 Biosynthesis
Zhaoyong Yang, Xiuling Chi, Masanori
Funabashi, Satoshi Baba, Koichi Nonaka,
Pallab Pahari, Jason Unrine, Jesse M.
Jacobsen, Gregory I. Elliott, Jürgen Rohr and
Steven G. Van Lanen
J. Biol. Chem. 2011, 286:7885-7892.
doi: 10.1074/jbc.M110.203562 originally published online January 7, 2011
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 10, pp. 7885–7892, March 11, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Characterization of LipL as a Non-heme, Fe(II)-dependent
␣-Ketoglutarate:UMP Dioxygenase That Generates
□
S
Uridine-5ꢀ-aldehyde during A-90289 Biosynthesis*
Received for publication, November 15, 2010, and in revised form, January 6, 2011 Published, JBC Papers in Press, January 7, 2011, DOI 10.1074/jbc.M110.203562
Zhaoyong Yang‡, Xiuling Chi‡, Masanori Funabashi§, Satoshi Baba§, Koichi Nonaka§, Pallab Pahari‡, Jason Unrine¶,
Jesse M. Jacobsen‡, Gregory I. Elliott‡, Ju¨rgen Rohr‡, and Steven G. Van Lanen‡1
From the ‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536,
§Biopharmaceutical Research Group I, Biopharmaceutical Technology Research Laboratories, Pharmaceutical Technology
Division, Daiichi Sankyo Co., Ltd., 389-4, Aza-ohtsurugi, Shimokawa, Izumi-machi, Iwaki-shi, Fukushima 971-8183, Japan, and the
¶Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, Lexington, Kentucky 40536
Fe(II)- and ␣-ketoglutarate (␣-KG)-dependent dioxygenases
are a large and diverse superfamily of mononuclear, non-heme
enzymes that perform a variety of oxidative transformations
typically coupling oxidative decarboxylation of ␣-KG with
hydroxylation of a prime substrate. The biosynthetic gene clus-
ters for several nucleoside antibiotics that contain a modified
uridine component, including the lipopeptidyl nucleoside
A-90289 from Streptomyces sp. SANK 60405, have recently been
reported, revealing a shared open reading frame with sequence
similarity to proteins annotated as ␣-KG:taurine dioxygenases
(TauD), a well characterized member of this dioxygenase super-
family. We now provide in vitro data to support the functional
assignment of LipL, the putative TauD enzyme from the
A-90289 gene cluster, as a non-heme, Fe(II)-dependent ␣-KG:
UMP dioxygenase that produces uridine-5ꢀ-aldehyde to initiate
the biosynthesis of the modified uridine component of A-90289.
The activity of LipL is shown to be dependent on Fe(II), ␣-KG,
and O2, stimulated by ascorbic acid, and inhibited by several
divalent metals. In the absence of the prime substrate UMP,
LipL is able to catalyze oxidative decarboxylation of ␣-KG,
although at a significantly reduced rate. The steady-state kinetic
parameters using optimized conditions were determined to be
based on structural differences: peptidyl nucleosides repre-
sented by pacidamycin from Streptomyces coeruleorubidus (2)
and mureidomycin from Streptomyces flavidovirens (3), lipodi-
saccharyl nucleosides represented by tunicamycins from Strep-
tomyces lysosuperificus (4), lipopeptidyl nucleosides repre-
sented by A-90289 from Streptomyces sp. SANK 60405 (5) and
caprazamycin from Streptomyces sp. MK730–62F2 (6), and
glycosyl-peptidyl nucleosides represented by the capuramycin-
type antibiotics A-500359s from Streptomyces griseus SANK
60196 (7), A-503083s from Streptomyces sp. SANK 62799 (8),
and A-102395 from Amycolatopsis sp. SANK 60206 (9). How-
ever, despite several notable structural variations, the last three
of these four groups share one signature feature that is critical
for biological activity: a uracil-containing nucleoside compo-
nent that consists of a high-carbon furanoside, wherein the
typical ribosyl moiety is replaced by a hexafuranoside (C6), hep-
tafuranoside (C7), or, in the case of tunicamycin, an undeca-
furanoside (C11) (Fig. 1) (10).
The biosynthetic gene clusters for the C7 nucleoside antibi-
otics A-90289 (5), liposidomycins (11), and caprazamycins (12)
have recently been reported. Interestingly, an open reading
frame (orf) encoding a protein with similarity to serine
hydroxymethyltransferases was uncovered in all of the gene
clusters. Serine hydroxymethyltransferases catalyze the inter-
conversion of Gly to Ser utilizing N5,N10-methylene tetrahy-
drofolate as a hydroxymethyl donor (13). Because the C7
nucleosides contain a C5Ј-glycyl unit (Fig. 1), this enzyme was
an obvious candidate to catalyze an aldol-type condensation
using uridine-5Ј-aldehyde as the “hydroxymethyl” donor in
place of N5,N10-methylene tetrahydrofolate. Rather unex-
pected was the uncovering of a homologous serine hydroxy-
methyltransferases within the biosynthetic gene clusters for the
C6 nucleosides A-500359 (14) and A-503083 (15), which are
characterized by a 5Ј-C-carbamoyl-uridine (Fig. 1). This dis-
covery suggested that a common mechanism is utilized to form
the high carbon scaffold, potentially through a common 5Ј-C-
glycyluridine intermediate, but subsequent tailoring step(s) are
required to eliminate a carbon unit during the biosynthesis of
the C6 nucleosides.
␣-KG
UMP
Km
؍ 7.5 ␮M, Km
؍ 14 ␮M, and kcat ≈ 80 min؊1. The
discovery of this new activity not only sets the stage to explore
the mechanism of LipL and related dioxygenases further but
also has critical implications for delineating the biosynthetic
pathway of several related nucleoside antibiotics.
Several nucleoside antibiotics from Streptomyces sp. and
related actinomycetes have been discovered that inhibit bacte-
rial translocase I (MraY) involved in the biosynthesis of pepti-
doglycan cell wall, an essential component for the survival of all
bacteria and a proven target for antibiotics (1). These nucleo-
side antibiotics have been classified into minimally four groups
* This work was supported in part by the Kentucky Science and Technology
Corporation (S. G. V. L.) and a National Science Foundation predoctoral fel-
lowship (J. J.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S18, Methods, and additional references.
Upon further comparative analysis of the identified gene
clusters, we noticed that only one additional orf was shared
among all of the putative biosynthetic genes. This orf encodes a
1 To whom correspondence should be addressed: 789 S. Limestone, Lexing-
ton, KY 40539. Tel.: 859-323-6271; Fax: 859-257-7547; E-mail: svanlanen@
uky.edu.
MARCH 11, 2011•VOLUME 286•NUMBER 10
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Characterization of ␣-KG:UMP Dioxygenase LipL
The realization that the product of both TauD and AtsK is an
aldehyde due to hydroxylation of a carbon bonded to a rela-
tively good leaving group led us to hypothesize that the shared
dioxygenase catalyzes a mechanistically similar reaction. Thus,
it was envisioned that the putative dioxygenase would function
prior to the serine hydroxymethyltransferases-like enzyme by
catalyzing the hydroxylation at C-5Ј of a uridine-related metab-
olite leading to a geminal diol or an unstable geminal-hydroxy
phosphoester that decomposes to phosphate and uridine-5Ј-
aldehyde. Alternatively, it could not be excluded a priori that
these newly discovered dioxygenases function as authentic tau-
rine dioxygenases to give aminoacetaldehyde that would serve
as an electrophile for an aldol-type condensation with an
unknown, modified uridine component. Therefore, it was of
general interest to elucidate the function of the TauD-like
dioxygenases as an initial step to delineating the biosynthetic
mechanism for high carbon nucleosides.
Using the hypothetical TauD (LipL) from the A-90289 bio-
synthetic gene cluster as a model enzyme, we now demonstrate
that uridine-5Ј-aldehyde is likely a shared, pathway intermedi-
ate by functionally assigning recombinant LipL as a non-heme,
Fe(II)-dependent ␣-KG:uridine-5Ј-monophosphate (UMP)
dioxygenase that catalyzes the formation of succinate, phos-
phate, and uridine-5Ј-aldehyde. The biochemical characteriza-
tion of LipL presented herein reveals a novel functional role for
a member of the Fe(II)- and ␣-KG-dependent dioxygenase
superfamily, setting the stage for in-depth mechanistic studies
on these new enzyme catalysts.
EXPERIMENTAL PROCEDURES
Chemicals and General Methods—5,5Ј-Dithiobis(2-nitro-
benzoic acid) (Ellman’s reagent), UMP disodium, ␣-KG sodium
salt, sodium succinate, sodium L-ascorbate, taurine, nucleo-
sides, and nucleotides were purchased from Sigma. Buffers and
salts, including Fe(II) chloride tetrahydrate, were purchased
from Fisher Scientific. Uridine-5Ј-aldehyde was synthesized
from uridine by protection of the 2Ј,3Ј-hydroxyl group with
p-methoxybenzaldehyde, and Pfitzner-Moffatt oxidation of the
resulting product provided 2Ј,3Ј-O-p-methoxybenzylideneuri-
dine-5Ј-aldehyde; the oxidized product was deprotected to
afford uridine-5Ј-aldehyde (see supplemental Methods and
supplemental Fig. S2) (20, 21). The SensoLyteꢁ MG Phosphate
Assay kit was purchased from AnaSpec (San Jose, CA). The
uncoupled reaction was analyzed using a succinate detection
kit from Megazyme International following the provided
instructions. UV/visible spectroscopy was performed with a
Bio-Tek ␮Quant microplate reader using MicrotestTM 96-well
plates (BD Biosciences) or a Shimadzu UV/visible-1800 spec-
trophotometer. Synthetic oligonucleotides were purchased
from Integrated DNA Technologies (Coralville, IA). DNA
sequencing was performed using the BigDyeTMTerminator
version 3.1 Cycle Sequencing kit from Applied Biosystems and
analyzed at the University of Kentucky Advanced Genetic
Technologies Center. HPLC was performed with a Waters Alli-
ance 2695 separation module (Milford, MA) equipped with a
Waters 2998 diode array detector and an analytical Apollo C-18
column (250 mm ϫ 4.6 mm, 5 ␮m) purchased from Grace
(Deerfield, IL). Electrospray ionization-MS was performed
FIGURE 1. Structures of representative nucleoside antibiotics that inhibit
bacterial translocase I. Highlighted in blue is the unusual C-C bond resulting
in the classification as high carbon nucleosides.
protein with sequence similarity to proteins annotated as ␣-ke-
toglutarate:taurine dioxygenases (TauD),2 which catalyze the
conversion of taurine to aminoacetaldehyde and sulfite (sup-
plemental Fig. S1) (16). TauD is the model member of a large
and diverse superfamily of mononuclear, non-heme Fe(II)- and
␣-ketoglutarate (␣-KG)-dependent enzymes that are generally
agreed to follow identical reaction coordinates involving the
oxidative decarboxylation of ␣-KG to give an enzyme-bound
Fe(IV)-oxo intermediate that is utilized to abstract a hydrogen
atom from a prime substrate, taurine for TauD, to yield a car-
bon-centered radical (17, 18). Typically, hydrogen abstraction
is followed by oxygen rebound of the Fe(III)-hydroxo species,
resulting in hydroxylation at this carbon, which for TauD
results in the formation of 1-hydroxy-2-aminoethanesulfonic
acid that decomposes to the products (16). The formation of an
aldehyde product is also observed with AtsK, a member of this
dioxygenase superfamily that catalyzes hydroxylation of an
alkyl sulfate ester that decomposes to sulfate and an aliphatic
aldehyde (supplemental Fig. S1B) (19).
2 The abbreviations used are: TauD, ␣-ketoglutarate:taurine dioxyge-
nases; ICP-MS, inductively coupled plasma mass spectrometric; ␣-KG,
␣-ketoglutarate.
7886 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of ␣-KG:UMP Dioxygenase LipL
using an Agilent 6120 Quadrupole MSD mass spectrometer 1200 series LC) with refractive index (Optilab rEX; Wyatt,
(Agilent Technologies, Santa Clara, CA) equipped with an Agi-
lent 1200 Series Quaternary LC system and an Eclipse XDB-
C18 column (250 mm ϫ 4.6 mm, 5 ␮m, 80 Å). NMR data were
collected using a Varian Unity Inova 500 MHz Spectrometer
(Varian, Inc., Palo Alto, CA).
Goleta, CA), multiangle laser light scattering (DAWN Heleos
III; Wyatt), and inductively coupled plasma mass spectrometric
(ICP-MS; Agilent 7500CX) detection. The refractive index
detector was used to determine protein concentration in the
eluting peaks assuming a dN/dC (differential refractive index)
value of 0.185, and the multiangle laser light scattering was used
in conjunction with the refractive index detector to verify the
molar masses of the eluting peaks using a partial Zimm model
(22). The ICP-MS was used as an element-specific detector to
identify V, Cr, Mn, Fe, Co, Ni, Cu, and Zn and to quantify Fe and
Zn content (23). Fe and Zn content were calibrated using
bovine hemoglobin and carbonic anhydrase as standards,
respectively. The mobile phase was 100 mM NH4NO3 at a flow
rate of 0.5 ml minϪ1 through a Superdex 200HR SEC column
(GE Healthcare).
Cloning and Expression of TauD and LipL—The genes were
amplified by PCR using the Expand Long Template PCR system
from Roche Applied Science with supplied buffer 2, 200 mM
dNTPs, 5% dimethyl sulfoxide, 10 ng of DNA template, 5 units
of DNA polymerase, and a 10 mM concentration of each of the
following primer pairs: EctauD (forward), 5Ј-GGTATTGAGG-
GTCGCatgagtgaacgtctgagcattac-3Ј/(reverse), 5Ј-AGAGGAG-
AGTTAGAGCCttaccccgcccgataaaacg-3Ј and lipL (forward),
5Ј-GGTATTGAGGGTCGCatgtccgtgctggggcgg-3Ј/(reverse),
5Ј-AGAGGAGAGTTAGAGCCtcatgagggcttcttcggtg-3Ј. DNA
templates for PCR cloning were Escherichia coli DH5␣
genomic DNA (tauD) and cosmid pN1 (lipL) (7). The PCR pro-
gram included an initial hold at 94 °C for 2 min, followed by 30
cycles of 94 °C for 10 s, 56 °C for 15 s, and 68 °C for 50 s. The
gel-purified PCR product was inserted into pET-30 Xa/LIC
using ligation-independent cloning as described in the pro-
vided protocol to yield pET30-EctauD and pET30-lipL, and the
genes were sequenced to confirm PCR fidelity.
Activity Assays Using HPLC—Reactions consisted of 50 mM
Tris-HCl (pH 7.5), 1 mM UMP, 1.25 mM ␣-KG, 200 ␮M ascor-
bate, 100 ␮M FeCl2, and 100 nM LipL at 30 °C. Reactions were
terminated by the addition of cold trichloroacetic acid to 5%
(w/v) or by ultrafiltration using a Microcon YM-3. Following
centrifugation to remove protein, the reaction was analyzed by
HPLC using a C-18 reversed-phase column and ion-pairing
conditions. A series of linear gradients was developed from 40
mM phosphoric acid-triethylamine pH 6.5 (A) to 90% acetoni-
trile (B) in the following manner (beginning time and ending
time with linear increase to % B): 0–8 min, 0% B; 8–18 min,
60% B; 18–25 min, 95% B; 25–32 min, 95% B; and 32–35 min,
0% B. The flow rate was 1.0 ml/min, and elution was monitored
at 260 nm. LC-MS was performed using a linear gradient from
0.1% formic acid in water to 0.1% formic acid in acetonitrile
over 20 min. The flow rate was 0.4 ml/min, and elution was
monitored at 254 nm.
Plasmids were introduced into E. coli BL21(DE3) cells, and
the transformed strains were grown in LB supplemented with
30 ␮g/ml kanamycin. Following inoculation of 500 ml of LB
with 30 ␮g/ml kanamycin, the cultures were grown at 18 °C
until the cell density reached an A600 ϭ 0.5 when expression
was induced with 0.1 mM isopropyl 1-thio-␤-D-galactopyrano-
side. Cells were harvested after an overnight incubation at 18 °C
and lysed using a French press with one pass at 15,000 p.s.i.
Following centrifugation, the protein was purified using affinity
chromatography with nickel-nitrilotriacetic acid-agarose from
Qiagen (Valencia, CA), and the recombinant proteins were
desalted into 50 mM Tris-HCl (pH 8), 100 mM NaCl, and 5%
glycerol using a PD-10 desalting column (GE Healthcare). The
purified protein was concentrated using an Amicon Ultra
10000 MWCO centrifugal filter (Millipore) and stored as glyc-
erol stocks (40%) at Ϫ20 °C. Protein purity was assessed by 12%
acrylamide SDS-PAGE; His6-tagged proteins were utilized
without further modifications.
Activity Assays by Phosphate Detection and Activity Optimiza-
tion—The activity of LipL was detected by UV/visible spectros-
copy by monitoring the formation of inorganic phosphate with
the malachite green binding assay (24). Routine reactions con-
sisted of 50 mM Tris-HCl (pH 7.5), 1 mM UMP, 1.25 mM ␣-KG,
100 ␮M ascorbate, 50 ␮M FeCl2, and 100 nM LipL. Optimal iron
concentration for activity was determined with assays contain-
ing 100 ␮M ascorbate, 40 nM LipL, and 0.1–750 ␮M FeCl2. After
identifying the optimal iron concentration, the optimal ascor-
bate concentration was determined with reactions containing
20 ␮M FeCl2, 30 nM LipL, and 0.5–50 mM ascorbate. All reac-
tions were carried out at 30 °C, and product formation was ana-
lyzed under initial velocity conditions.
␣-KG:Taurine Dioxygenase Activity—Standard reactions
consisted of 50 mM Tris-HCl (pH 7.5), 0.5 mM taurine, 1 mM
␣-KG, 0.2 mM ascorbate, 0.1 mM FeCl2, and 20 ␮g/ml LipL or
2.5 ␮g/ml EcTauD at 30 °C (16). Control reactions were per-
formed without the addition of either ␣-KG or enzyme. At
desired time points, 80 ␮l of the reaction mixture was added to
20 ␮l of 0.25 M EDTA and 10 ␮l of Ellman’s reagent (1 mg/ml in
0.1 M phosphate (pH 7.0)). The mixture was incubated at room
temperature for 5 min prior to obtaining the absorbance at 415
nm. Sodium sulfite was used for calibration curves yielding a
linear response from 25 to 400 ␮M sulfite. The specific activity
of EcTauD, calculated from end point assays under initial veloc-
ity conditions, was determined using the reaction mixture
above except with 0.3 ␮g/ml EcTauD.
Kinetic Analysis—Assays consisted of 50 mM Tris-HCl (pH
7.5), 1 mM ascorbate, 20 ␮M FeCl2, LipL, and near saturating
UMP (2 mM) with variable ␣-KG (5 ␮M–3 mM) or near saturat-
ing ␣-KG (1 mM) and variable UMP (2 ␮M–1 mM). The reaction
was performed at 30 °C for 3 min and analyzed under initial
velocity conditions. Product formation was determined using
either the malachite green binding assay or HPLC. Each data
point represents a minimum of two (HPLC assay) or three (mal-
achite green binding assay) replicate end point assays; kinetic
constants were obtained by nonlinear regression analysis using
Metal Content—Iron and zinc content in the proteins was
determined using size exclusion chromatography (Agilent, GraphPad Prism (GraphPad Software, La Jolla, CA).
MARCH 11, 2011•VOLUME 286•NUMBER 10
JOURNAL OF BIOLOGICAL CHEMISTRY 7887

Characterization of ␣-KG:UMP Dioxygenase LipL
Dependence of the Reaction on O2 and Fate of Oxygen Atoms—
A 1-ml reaction containing 10 mM Tris-HCl (pH 8.0), 1 mM
UMP, 1.5 mM ␣-KG, 1 mM fresh ascorbate, and 100 ␮M fresh
FeCl2 was placed under a N2 atmosphere and purged twice by
bubbling in N2 for 10 min. The atmosphere was maintained in
N2 or exchanged with 16O2 (control) or 18O2, and the reaction
was initiated by the addition of 2.5 ␮l of LipL (final concentra-
tion of 1 ␮M). The reaction was incubated at room temperature
in a hermetically sealed flask for 90 min. The reaction was ter-
minated by the addition of cold trichloroacetic acid (5%) and
the components analyzed by ion pairing chromatograph and
LC-MS as described above.
RESULTS
Bioinformatics Analysis—BLAST analysis of LipL revealed
closest sequence similarity to proteins primarily annotated as
␣-KG:taurine dioxygenases (22–28% identity). Sequence simi-
larity of LipL to E. coli TauD, a well characterized ␣-KG:taurine
dioxygenase for which the structure is known (25), is signifi-
cantly less (15% identity/24% similarity). Similarly, low levels of
sequence identity were also found with other structurally elu-
cidated dioxygenases of this superfamily, such as AtsK (19) and
clavaminic acid synthase from Streptomyces clavuligerus (26).
All members of this dioxygenase superfamily contain an
essential Fe(II)-binding motif HX(D/E)XnH, where X is any
amino acid (18). The iron-binding motif of LipL was putatively
assigned by alignments with EcTauD, which revealed the
FIGURE 2. Identification of the LipL product. HPLC analysis of the LipL reac-
tion using authentic UMP without ␣-KG (I), a 15-min reaction containing all of
the necessary components (II), the 15-min reaction mixture spiked with syn-
thetic uridine-5Ј-aldehyde (III), and synthetic uridine-5Ј-aldehdye (IV) are
shown. A260, absorbance at 260 nm.
potential triad within LipL (His123, Asp125, and His266
)
Ϫ
(supplemental Fig. S3). In addition to the three conserved met- peak had an (MϪH) ion at m/z ϭ 240.8, consistent with the
Ϫ
al-binding residues, an Arg residue involved in ␣-KG binding is molecular formula for uridine-5Ј-aldehyde (expected (MϪH)
also conserved (Arg279 for LipL). Thus, based on sequence anal- ion at m/z ϭ 241.1) (supplemental Fig. S6). In contrast, only
ysis, LipL apparently contains the essential residues to function substrates UMP and ␣-KG were observed by LC-MS when the
as an Fe(II)- and ␣-KG-dependent dioxygenase (17–19), and enzyme was omitted (supplemental Fig. S7). To confirm the
these residues are conserved in the other TauD-like enzymes identity of the new peak, uridine-5Ј-aldehyde was synthesized
from the A-500359, A-503083, liposidomycin, and caprazamy- and confirmed by 1H NMR and 13C NMR spectroscopy (sup-
cin gene clusters (supplemental Fig. S3).
plemental Fig. S2). HPLC and LC-MS analysis with co-injec-
Functional Assignment and Assay Development—LipL was tions of authentic material, which was likewise previously dem-
cloned and expressed in E. coli BL21(DE3), and the recombi- onstrated to be unstable upon several column purification
nant protein was purified to near homogeneity as judged by methods (21), revealed uridine-5Ј-aldehyde was indeed the
SDS-PAGE (supplemental Fig. S4A). Given the closest se- product of LipL (Fig. 2).
quence similarity to proteins annotated as TauD, the tauD gene
TauD and related Fe(II)- and ␣-KG-dependent dioxygenases
from E. coli was also cloned, and the recombinant protein was produce CO2 and succinate upon oxidative decarboxylation of
purified to serve as a control (supplemental Fig. S4B). Using ␣-KG (16–18). Succinate was confirmed as a product for LipL
Ellman’s reagent for spectroscopic detection, recombinant by utilizing LC-MS with commercial succinate as a control
EcTauD catalyzed the expected ␣-KG- and taurine-dependent (supplemental Fig. S6). Alternatively, an enzymatic coupled
formation of sulfite with a specific activity of 1.4 mmol/min per assay was used to confirm that succinate was produced during
mg, which is nearly identical to the reported value (supplemen- the LipL-catalyzed reaction (supplemental Fig. S8) (27). Analo-
tal Fig. S5) (16). In contrast, no reaction was observed when gous to sulfite production by TauD, phosphate was also colori-
LipL was incubated under the identical conditions, consistent metrically detected by employing a malachite green binding
with LipL having a unique activity.
Activity of LipL was next tested with alternative prime sub- mation of succinate, phosphate, and uridine-5Ј-aldehyde.
strates in place of taurine. When the enzyme was incubated Activity Optimization, Specificity, and General Characteriza-
assay (24). Thus, LipL was directly shown to catalyze the for-
with UMP, a new peak was observed by HPLC with concomi- tion—The requirements for LipL activity was assessed by HPLC
tant loss in the peak corresponding to UMP (Fig. 2). The new analysis following uridine-5Ј-aldehyde formation and colori-
compound was unstable upon column purification under a metric analysis by detecting phosphate formation, with the lat-
variety of conditions; however, LC-MS analysis of the crude ter assay utilized for analysis under initial velocity conditions.
reaction mixture consisting of ϳ95% conversion of UMP to The activity was absolutely dependent upon ␣-KG and O2 (Fig.
product (as determined by ion-pairing HPLC) revealed the new 2 and supplemental Fig. S9). The addition of Fe(II) or ascorbate
7888 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 10•MARCH 11, 2011

Characterization of ␣-KG:UMP Dioxygenase LipL
FIGURE 3. Activity optimization and kinetic analysis of LipL using the malachite green binding assay. A, optimal activity of LipL with respect to varied
FeCl2 in reactions containing 100 ␮M ascorbate and 40 nM LipL. B, optimal activity of LipL with respect to varied ascorbate in reactions containing of 20 ␮M FeCl2
and 30 nM LipL. C, single-substrate kinetic analysis using variable ␣-KG (2 ␮M–3 mM), near saturating UMP (2 mM) and 34 nM LipL. D, single-substrate kinetic
analysis using variable UMP (2 ␮M–1 mM), near saturating ␣-KG (1 mM) and 34 nM LipL. Data represent the average of minimally three independent replicates
obtained using end point assays under initial velocity conditions; S.E. was Ͻ15% in all cases.
was not essential for activity; however, inclusion of EDTA abol- has been reported for TauD (16) and clavaminic acid synthase
ished activity, suggesting that Fe(II) is required (supplemental (29).
Fig. S10). Subsequently, ICP-MS analysis revealed LipL co-pu-
Kinetic Analysis—Steady-state kinetic analysis was per-
rified with 14 Ϯ 2% iron (mol/mol), thus explaining the residual formed using both the malachite green binding assay to detect
activity in the absence of additional Fe(II). LipL was found to phosphate and HPLC to detect uridine-5Ј-aldehyde. Single-
have optimal activity in the range of 2–100 ␮M FeCl2 (Fig. 3A). substrate kinetic experiments with phosphate detection
Using 20 ␮M FeCl2, optimal activity with respect to ascorbate revealed typical Michaelis-Menten kinetics, yielding kinetic
was determined to be 1 mM for LipL (Fig. 3B).
constants of Km ϭ 7.5 Ϯ 2.4 ␮M and kcat ϭ 92 Ϯ 9 minϪ1 with
The specificity of LipL toward different nucleosides and respect to ␣-KG and Km ϭ 14 Ϯ 3 ␮M, kcat ϭ 76 Ϯ 8 minϪ1 with
nucleotides was next analyzed. Uridine and UDP were not rec- respect to UMP (Fig. 3, C and D). Although detection limits
ognized as substrates, demonstrating that LipL is specific for precluded a more accurate kinetic analysis, similar plots and
the monophosphorylated nucleoside (supplemental Fig. S11). extracted kinetic constants were obtained using HPLC detec-
LipL was unable to catalyze dephosphorylation and aldehyde tion (supplemental Fig. S13).
formation using the remaining canonical, monophosphory-
Uncoupled Oxidative Decarboxylation of ␣-KG—Many
lated ribo- or deoxyribonucleotides (data not shown). Similar to enzymes of the ␣-KG-dependent dioxygenase superfamily
the high specificity toward the prime substrate UMP, pyruvate, including TauD catalyze oxidative decarboxylation of ␣-KG
␣-ketoadipate, ␣-ketobutyrate, ␣-ketovalerate, and oxaloace- without the inclusion of the prime substrate (18, 27, 30). Utiliz-
tate could not substitute for ␣-KG in the reaction.
ing LC-MS for detection, succinate was similarly observed for
Finally, the activity with different metals was tested. Potas- LipL without including UMP in the reaction (supplemental Fig.
sium ions, Fe(III), or divalent metals including Mg2ϩ, Ca2ϩ, and S14). The enzyme-coupled assay to detect succinate production
third-row transition metals were not able to replace Fe(II) or was employed to confirm the LC-MS result (supplemental Fig.
increase the specific activity when included in the reaction with S8), and kinetic analysis with UMP yielded nearly an identical
FeCl2 (supplemental Fig. S12). Instead, several metals inhibited
k
cat (78 minϪ1) relative to kinetic analysis by detection of phos-
the reaction including Zn2ϩ, which was the only metal in addi- phate or uridine-5Ј-aldehyde. In contrast, the rate of succinate
tion to Fe that was found to co-purify with LipL (19 Ϯ 2% mol/ formation by LipL was significantly reduced in the absence of
Ϫ1
mol) based on ICP-MS analysis. Further analysis revealed that UMP (supplemental Fig. S8), yielding a kcat ϭ 0.4 min for
Zn2ϩ strongly inhibited LipL activity (supplemental Fig. S12), LipL, which corresponds to a relative turnover of 0.5% com-
and therefore co-purification with Zn2ϩ is likely due to adven- pared with the UMP-containing reaction.
titious binding as observed with other non-heme, iron-depen-
Although succinate was readily detected, no other hypothet-
dent oxygenases (28). Inhibition by divalent transition metals ical reaction intermediates (for example, uridine) or shunt
MARCH 11, 2011•VOLUME 286•NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7889

Characterization of ␣-KG:UMP Dioxygenase LipL
FIGURE 4. Proposed biochemical pathway leading to several high carbon nucleosides inhibiting bacterial translocase I. Highlighted in bold is the
enzyme characterized in this study; enzymes with likely identical function as LipL include ORF7 from the A-500359 gene cluster, CapA from the A-503083 gene
cluster, LpmM from the liposidomycin gene cluster, Cprz15 from the caprazamycin gene cluster, and PacF/PacX from the pacidamycin gene cluster. AMT,
aminotransferase; GTase, glycosyltransferase.
products were detected by HPLC with photodiode array or MS dent dioxygenase superfamily. We have characterized a repre-
detection. Furthermore, hydrogen peroxide was not detected
using an enzyme-coupled reaction with horseradish peroxidase
(31). In addition, UMP or uridine was not detected when the
reverse reaction was assayed.
sentative enzyme from the A-90289 gene cluster to reveal yet
another unusual reaction catalyzed by enzymes of the dioxyge-
nase superfamily: the net dephosphorylation and two-electron
oxidation of UMP. Thus, during the biosynthesis of C7 high
carbon nucleosides, the modified nucleoside can now be
described to begin with the conversion of UMP to uridine-5Ј-
aldehyde (Fig. 4), results that are consistent with prior feeding
experiments with other model high carbon nucleoside antibi-
otics (37–39). The uridine-5Ј-aldehyde intermediate is subse-
quently condensed with glycine to form C-5Ј-glycyluridine,
which remains intact during the formation of the lipopeptidyl
nucleosides. The identification of a homologous enzyme within
the A-500359 and A-503083 gene clusters suggest that an iden-
tical reaction occurs during the biosynthesis of C6 high carbon
nucleosides, although in this case the resulting C-5Ј-glycyluri-
dine undergoes oxidative decarboxylation catalyzed by a dis-
tinct, unidentified enzyme to yield the final nucleoside (Fig. 4).
The lipopeptidyl nucleosides contain an additional aminoribo-
syl moiety, and the pathway to this component is proposed to
also proceed through uridine-5Ј-aldehyde, which is diverted
into this alternative pathway upon transamination, and subse-
quent processing yields the activated NDP-amino sugar for
attachment to the aglycon. In addition, the recent identification
of the biosynthetic gene cluster for pacidamycin has revealed a
homologous dioxygenase and aminotransferase, suggesting
that the unusual enamide-containing nucleoside originates by
the tandem action of these two enzymes (40, 41). Thus, this
newly discovered enzyme activity is likely essential for the bio-
synthesis of a number of structurally distinct nucleoside
antibiotics.
Isotope Enrichment Studies—The fate of oxygen atoms
derived from O2 was examined. After saturating a reaction mix-
ture with 18O2 (97 atom %), excess LipL was added, and the
reaction was allowed to proceed for 90 min under an 18O2
atmosphere at room temperature. Detection by LC-MS
Ϫ
revealed 97% of the total succinate had an (MϪH) ion at
m/z ϭ 119.0, consistent with the incorporation of one atom of
Ϫ
oxygen 18O2 (expected (MϪH) ion at m/z ϭ 119.0) (supple-
mental Fig. S15). Thus, incorporation into succinate from O2
was essentially 100%. When the reaction was performed in
H218O (final of 80 atom %), detection by LC-MS revealed mass
peaks for succinate that correspond to ϳ79% incorporation of
an oxygen atom from H218O and ϳ83% incorporation for the
uncoupled reaction. When LC was bypassed for direct analysis
of the uncoupled reaction, the mass corresponding to residual
␣-KG revealed 75% 18O incorporation, an expected result due
to nonenzymatic exchange of the ␣-keto acid (32). Unfortu-
nately, the rapid and reversible formation of a geminal diol in
water did not allow the analysis of putative oxygen incorpora-
tion into uridine-5Ј-aldehyde.
DISCUSSION
Enzymes of the Fe(II)- and ␣-KG-dependent dioxygenase
superfamily catalyze a diverse array of biotransformations in
natural product biosynthesis, including hydroxylation of Arg in
viomycin biosynthesis (33, 34); O-demethylation in morphine
biosynthesis (35); and hydroxylation, cyclization, and desatura-
tion by clavaminic acid synthase in the biosynthesis of the
␤-lactam clavulonic acid (29, 36). Bioinformatics analysis of
several recently uncovered gene clusters for minimally two dif-
ferent families of nucleoside antibiotics revealed a shared open
reading frame encoding a protein with low sequence similarity
Most of the biochemical characteristics for LipL have been
reported for other enzymes of the Fe(II)- and ␣-KG-dependent
dioxygenase superfamily. For instance, activity is stimulated by
ascorbate, certain divalent metals inhibit activity, activity is
specific for both the prime substrate and ␣-KG, and oxidative
to TauD, the best studied enzyme of Fe(II)- and ␣-KG-depen- decarboxylation of ␣-KG still occurs in the absence of the prime
7890 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 10•MARCH 11, 2011

Characterization of ␣-KG:UMP Dioxygenase LipL
4. Tamura, G., Sasaki, T., Matsuhashi, M., Takatsuki, A., and Yamasaki, M.
(1976) Agric. Biol. Chem. 40, 447–449
substrate, all characteristics realized for TauD and AtsK,
among others (18). Clearly, the unique feature of LipL is the
specific utilization of UMP and the mechanism by which the
net dephosphorylation and oxidation occurs. Previously, it was
logically speculated that this transformation occurs via tandem
reactions catalyzed by a phosphatase and oxidoreductase.
Common catalytic strategies for phosphatases include (i) for-
mation of a phosphoenzyme intermediate utilizing a conserved
Cys as the nucleophile or (ii) the use of a binuclear center con-
sisting of Mg2ϩ, Mn2ϩ, Fe2ϩ, and/or Zn2ϩ(42). However, our
cumulative biochemical data suggest that LipL catalyzes
dephosphorylation via a cryptic hydroxylation germinal to a
good leaving group and thus likely employs the prototypical
Fe(II)- and ␣-KG-dependent dioxygenase mechanism reminis-
cent of TauD and AtsK, wherein decarboxylative oxidation of
␣-KG is coupled to the formation of an Fe(IV)-oxo intermedi-
ate that serves as the strong oxidizing agent for hydrogen
abstraction of the prime substrate (supplemental Fig. S16).
Alternatively, a few additional mechanisms could be envisioned
for LipL, including sequential hydrogen abstractions at C-5Ј
and C-4Ј of UMP analogous to the proposed desaturation
mechanism of certain Fe(II)- and ␣-KG-dependent dioxyge-
nases (18), which in this case would be followed by phosphate
hydrolysis and enol tautomerization to yield the final product
(supplemental Fig. S17). A clear distinction between hydroxy-
lation versus dephosphorylation/oxidation mechanisms is the
fate of the bridging phosphoester oxygen, and experiments are
under way to examine this feature of the reaction.
5. Funabashi, M., Baba, S., Nonaka, K., Hosobuchi, M., Fujita, Y., Shibata, T.,
and Van Lanen, S. G. (2010) ChemBioChem 11, 184–190
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The cumulative data suggest that LipL and by extension the
homologous enzymes involved in the biosynthesis of C6 and C7
high carbon nucleosides are new members of the superfamily of
Fe(II)- and ␣-KG-dependent dioxygenases, once again high-
lighting the versatility and significance of these biocatalysts in
nature. Interestingly, enzymatic phosphate release as a mecha-
nism of phosphate salvage analogous to sulfite and sulfate for-
mation for TauD and AtsK, respectively, has been prognosti-
cated as a potential function for this dioxygenase superfamily
(18), and we have demonstrated that this is indeed possible by
providing the enzyme precedence for this chemistry. It is also
foreseeable that the biosynthesis of other high carbon nucleo-
side antibiotics such as the uracil-containing tunicamycin (10)
and A-94964 (43), the cytosine-containing ezomycin A1 (44),
and the adenine-containing griseolic acid (45) and amipurimy-
cin (46) employ a similar enzymatic strategy for assembling a
high carbon sugar skeleton (supplemental Fig. S18). Regardless
of these possibilities, we have clearly demonstrated a novel
enzyme function that has significance for the biosynthesis of
several nucleoside antibiotics, which now sets the stage to inter-
rogate the mechanism of the newfound dioxygenase family and
delineate downstream catalytic events in the biosynthesis of
several compounds of potential therapeutic significance.
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