Comparative genomics guided discovery of two missing archaeal enzyme families involved in the biosynthesis of the pterin moiety of tetrahydromethanopterin and tetrahydrofolate

Article abstract of DOI:10.1021/cb300342u

C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H₄-MPT) or tetrahydrofolate (H₄-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin dip

Full text of DOI:10.1021/cb300342u

Letters
pubs.acs.org/acschemicalbiology
Comparative Genomics Guided Discovery of Two Missing Archaeal
Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of
Tetrahydromethanopterin and Tetrahydrofolate
,
†
†
⊥
‡
‡
†
§
Valer
́ ́
ie de Crecy-Lagard,* Gabriela Phillips, Laura L. Grochowski, Basma El Yacoubi, Francis Jenney,
∥
Michael W. W. Adams, Alexey G. Murzin, and Robert H. White
†Department of Microbiology and Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville,
Florida 32611-0700, United States
‡
Department of Biochemistry (0308), Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States
§Department of Basic Sciences, Georgia Campus, Philadelphia College of Osteopathic Medicine, Suwanee, Georgia 30024, United
States
∥
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States
⊥MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
*
S Supporting Information
ABSTRACT: C-1 carriers are essential cofactors in all domains of life, and in
Archaea, these can be derivatives of tetrahydromethanopterin (H -MPT) or
4
tetrahydrofolate (H -folate). Their synthesis requires 6-hydroxymethyl-7,8-
4
dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of
pathways that lead to its formation were unknown until the recent discovery of
the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the
conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-
dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem.,
2
81, 37586−37593 and Grochowski, L. L.; et al. (2007) Biochemistry 46, 6658−
6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show
that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of
the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of
the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these
missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar
architectures perform the same biochemical function.
1
The availability of over 3000 published genome sequences has
enabled the use of comparative genomic approaches to drive
Most organisms use H -folate (Figure 1) as the essential
4
carrier of C fragments in both anabolic and catabolic reactions.
1
2
,3
the biological function discovery process. Classically, one
used to link a gene with function by genetic or biochemical
approaches, a lengthy process that often took years.
Phylogenetic distribution profiles, physical clustering, gene
fusion, coexpression profiles, structural information and other
genomic or postgenomic derived associations can be now used
to make very strong functional hypotheses that can then be
The known exceptions are the methanogenic Archaea that use
7
H -MPT (Figure 1) and methylotrophic bacteria that use
4
8
dephospho-H
-MPT. The situation in Archaea is quite diverse.
4
Halophilic Archaea such as Halobacterium species harbor
9
folates. Hyperthermophiles like Pyrobaculum or Sulfolobus
species use C -carriers lacking the C-7 methyl group on the
1
10
pterin as seen in methanopterin. Methanogenic Archaea such
4
,5
quickly validated by simple genetic and/or biochemical tests.
as Methanobacterium thermoautotrophicum ΔH (now called
The whole procedure can occur in just weeks, taking advantage
of the constantly growing available postgenomic resources such
Methanobacterium thermoautotrophicus) use H -MPT, whereas
4
11
Thermococcus litoralis and Pyrococcus furiosus use only a more
exotic derivative of methanopterin containing poly-β-(1→4)-N-
5
as gene deletion or expression libraries. Here, we illustrate this
paradigm shift with the discovery of two archaeal protein
families involved in the synthesis of 6-hydroxymethyl-7,8-
dihydropterin diphosphate (6-HMDP), the precursor of the
acetylglucosamine as side chains on their C
1
-carrier coen-
12−14
zyme.
contain both H
Certain Archaea such as Methanosarcina barkeri
15
-MPT and H -folate derivatives. Sulfolobus
4
4
pterin containing moiety of the essential C -carriers tetrahy-
1
drofolate (H -folate) and tetrahydromethanopterin (H -MPT)
Received: April 18, 2012
Accepted: August 29, 2012
Published: August 29, 2012
4
4
(
Figure 1). These enzymes had eluded classical genetic and
6
biochemical approaches and had been missing for decades.
©
2012 American Chemical Society
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Figure 1. Early steps of tetrahydrofolate and tetrahydromethanopterin pathways in Bacteria and Archaea. Most bacteria use the FolE (or FolE2)/
FolB/FolK route (in blue) to 6-HMDP even if some use the bacterial PTPS-III shunt (in green). Several routes to the common 6-HMDP
intermediate in tetrahydrofolate and tetrahydromethanopterin are found in Archaea. A common pathway is the FolE2/MptD/MptE route (in red)
such as in H. volcanii paralleling the bacterial pathway. However, some methanogens such as M. jannaschii use the MptA/MptB/MptD/MptE route,
whereas P. furiosus uses the archaeal PTPS-III shunt. Phosphatases still to be identified are noted by a question mark (?). FolE/FolE2, GTP
cyclohydrolase IA/IB (GCYH-IA/B); FolB, 7,8-dihydroneopterin aldolase (DHNA); FolK, 7,8-dihydro-6-hydroxymethylpterin diphosphokinase (6-
HMDPK); MptA, archaeal GTP cyclohydrolase I (Fe(II)-dependent enzyme); MptB, Fe(II) dependent-cyclic phosphodiesterase; MptD, archaeal
specific DHNA; MptE, archaeal specific 6-HMDPK; PTPS-III/PTPS-V/PTPS-VI, pyruvoyltetrahydropterin synthase paralogs involved in 6-HMDP
synthesis.
22−24
solfataricus contains a hybrid coenzyme C -carrier coenzyme
to form 6-HMD
(Figure 1). In all cases, 6-HMD is then
1
harboring a nonmethylated pterin and the same arylamine
moiety found in methanopterin. Although numerous
diphosphorylated with ATP by a 7,8-dihydro-6-hydroxyme-
1
6
thylpterin diphosphokinase (6-HMDPK), encoded in E. coli by
25
variations in the C -carrier structures exist among the various
folK to form 6-HMDP.
1
archaeal lineages, the early steps in the syntheses of H -folate
Methanocaldococcus jannaschii was the first Archaea with a
sequenced genome. It was immediately apparent that this
organism lacked homologues of FolE, FolB, and FolK and used
4
and of H -MPT and its derivatives, leading to the formation of
4
17
the 6-HMDP intermediate, have been predicted to be similar
Figure 1). The 6-HMDP pathway is well characterized in
26
(
nonorthologous enzymes to catalyze the same reactions. This
prediction was confirmed as more archaeal genomes became
available (Figure 2). As shown in Figure 2, a minority of
Archaea (16 out of 58 analyzed) contained homologues of the
canonical FolE and expression of the corresponding gene from
Sulfolobus solfataricus P2 (sso0364) complemented the deoxy-
bacteria, plants, and fungi. GTP cyclohydrolase IA (GCYH-IA
or FolE) or GTP cyclohydrolase IB (GCYH-IB or FolE2)
catalyze the first step of the pathway producing 7,8-
1
8−20
dihydroneopterin triphosphate (H NTP) from GTP.
2
H NTP produces 7,8-dihydroneopterin (H Neo) after the
2
2
27
lost of a diphosphate and a phosphate. Then, 7,8-
thymidine (dT) auxotrophy of an E. coli ΔfolE mutant. Most
Archaea (40/58 analyzed) contained homologues of the more
recently discovered FolE2 (Figure 2) that were experimentally
validated in a few species. The folE2 mutant of Haloferax
dihydroneopterin aldolase (DHNA) encoded in Escherichia
2
1
coli by folB catalyzes the formation of 6-hydroxymethyl-7,8-
dihydropterin (6-HMD) from H Neo. A derivation from the
2
2
8
classical bacterial 6-HMDP synthesis pathway occurs in
Plasmodium falciparum and various bacteria. The DHNA step
volcanii (ΔHVO_2348) is a dT and hypoxanthine auxotroph,
and the M. jannaschii FolE2 homologue MptA (MJ0775) is a
is bypassed by PTPS-III that cleaves the side chain of H NTP
unique Fe(II)-dependent GTP cyclohydrolase IB that forms
2
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Figure 2. Phylogenetic distribution of predicted 6-HMDP synthesis genes in a subset of archaeal genomes. The presence of a symbol denotes the
presence of a member of the protein family represented in that specific column in the genome covered in the corresponding line. Symbols and
corresponding protein family are in the same color. Abbreviations have been defined in the Figure 1 legend. 6-HMDP synthesis genes might still be
unidentified in organisms highlighted in yellow. Organisms highlighted in beige are most certainly pterin auxotrophs. Symbols linked by a line
represent fused proteins. The full analysis is available in the Public SEED database in the Subsystem: “Pterin Biosynthesis Archaea”.
2
9
7
,8-dihydroneopterin 2′,3′-cyclic phosphate. In M. jannaschii,
(Figure 2) that certainly derive from a lateral gene transfer
event (the closest homologue to these two proteins is the fused
FolKB from Pneumocystis carinii f. sp. macacae (AAN38834.1)
with a Blastp E-value of 9e-40). A few Archaea such as P.
furiosus or Methanosarcina barkeri str. fusaro harbor proteins of
the PTPS-III family (Figure 2) that in bacteria function in a
MptB (MJ0837), a cyclic phosphodiesterase, is required to
cleave the cyclic phosphate to form a mix of 7,8-
dihydroneopterin 2′-monophosphate and 7,8-dihydroneopterin
30
3
′-monophosphate. This pathway, involving a 7,8-dihydro-
neopterin 2′,3′-cyclic phosphate intermediate, could be specific
to a subset of methanogens because homologues of MptB are
mostly found in Methanococcales (Figure 2). Even if the first
archaeal 6-HMDP biosynthesis enzymes have been charac-
terized, the remaining steps encoded in bacteria by FolB and
FolK remain to be discovered in most Archaea. The
identification and characterization of these missing gene
families is the focus of this study.
DHNA bypass where H NTP is converted directly to 6-
2
22−24
HMDP
(Figure 1). Surprisingly, in vitro, the PTPS-III
homologue from P. furiosus, PF1278, catalyzed the cleavage of
7,8-dihydroneopterin monophosphate (H NMP) to 6-HMD,
2
but H NTP was not a substrate (Supporting Information and
2
Figure 1). Finally, we recently showed that close homologues of
PTPS-III with a slightly different active site motif named PTPS-
31
Only two sequenced Archaea (Sulfolobus acidocaldarius DSM
39 and Caldivirga maquilingensis IC-167) contain homologues
of bacterial FolB proteins fused with homologues of bacterial
FolK proteins (Saci_1101 and Cmaq_0517, respectively)
VI were found in a few Sulfolobus species (Figure 2).
Expressing the PTPS-VI gene from S. solfataricus (sso2412)
partially complemented the dT auxotrophy of a ΔfolB E. coli
6
31
mutant suggesting a role of PTPS-VI proteins in 6-HMDP
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Figure 3. Comparative genomic evidence. (A) Clustering of COG1634 and COG2098 genes with pterin and cofactor biosynthetic related genes.
Abbreviation not found in the text: FolP-like, dihydropteroate synthase-like enzyme homologous to the bacterial folate enzyme FolP but of unknown
5
9
60
61
function; MptG, β-ribofuranosylaminobenzene 5′-phosphate synthase; FolM, alternative dihydrofolate reductase; F420-lig, coenzyme F420-0:
62
L-glutamate ligase. (B) The archaeal DHNA (MptD) tetramer with bound pterin ring mimic (PDB 2OGF). The individual subunits of MJ0408 are
shown with differently colored cartoons, the bound ligand 8-oxoguanine with orange carbons. (C) Putative active site of the archaeal DHNA with
manually docked neopterin. The MptD structure is from PDB 2IEC, and the neopterin ligand (orange carbons) is from PDB 2O90 (in alternative
conformation B). The active site residues contributed by three different subunits are shown with green, cyan, and magenta carbons, respectively (as
in panel B and Supplemental Figure 3).
3
9
synthesis even if the substrate specificity of this family is yet to
be experimentally determined (Figure 1). In summary, 56 out
of 58 of the archaeal genomes analyzed lacked a FolK
homologue, and 47 out of 58 lacked a FolB, PTPS-III, or
PTPS-VI homologue. Hence, we set out to identify these
missing archaeal 6-HMDP synthesis enzymes using a
combination of comparative genomic approaches.
structures of bacterial GST-II-like sialyltransferases (Supple-
mental Figure 1). These bacterial enzymes use CMP-NeuAc as
a sugar donor and have a distinct fold that is also found in
40
mammalian sialyltransferases. Comparison of the sialyltrans-
ferase and TPK structures revealed a common structural core
and similar binding modes of their respective products, CMP
and AMP, suggesting a distant evolutionary relationship of
these protein families (Supplemental Figure 2). The COG1634
members are predicted to share the NMP-binding site
We first searched for genes that physically clustered with
pterin related genes using the clustering tool of the SEED
2
platform and identified the COG1634/DUF115 gene family as
(
Supplemental Figure 1). In addition, COG1634 and TPK
a candidate (Figure 3A). Members of COG1634 are
uncharacterized proteins found in most Archaea (Figure 2)
and are part of the thiamin pyrophosphokinase (TPK, thiamin
family members share the metal (Mg(II)) ion-binding site,
involved in binding and transfer of the diphosphate group
(Supplemental Figures 1 and 2). On the basis of the physical
clustering evidence and fold homology, we predicted that
COG1634 was the missing archaeal 6-HMDPK family. The
homology with the sialyltransferase family opens the possibility
that members of the COG1634 family may utilize other
nucleoside triphosphates, e.g., CTP. There are documented
cases in archaeal biosynthetic pathways where CTP substitutes
for ATP; for example, the Archaeon-specific riboflavin kinase
32
diphosphokinase) catalytic domain superfamily. TPK is a
thiamin salvage enzyme that transfers the diphosphate group of
ATP to thiamin to form thiamin diphosphate, the active form
3
3
of the cofactor. TPK consists of two domains: the N-terminal
catalytic domain that binds ATP and the C-terminal substrate-
34−37
binding domain that binds thiamin.
The COG1634 family
members show sequence similarity to the TPK catalytic domain
but not to the C-terminal domain (Supplemental Figure 1).
38
41
Moreover, fold recognition servers, e.g., FFAS, predict the
TPK catalytic domain being a good template for the COG1634
subunit fold; they also suggest even higher-scoring hits to the
uses CTP as its phosphoryl donor, and the archaeal FAD
synthetase (RibL) catalyzes the cytidylation of FMN with
42
CTP.
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R
Figure 4. Experimental validations. (A) The MG1655 ΔfolB::Kan strain (VDC3276) was transformed with the empty vector pBAD24 (top), pfolB
Ec
(
middle), or pMJ0408 (bottom). The resulting strains were plated on LB (with appropriate antibiotics), LB supplemented with arabinose (0.2%), or
LB supplemented with dT and grown for 48 h. (B) The C600 ΔfolK::tetB strain was transformed with empty plasmid pBAD24 (top), pfolKEc
middle), or pMJ1634 (bottom). The resulting strains were plated on LB (with appropriate antibiotics) with or without dT supplementation and
45
(
grown for 48 h. In both cases, complementation of the dT auxotrophy phenotypes by the archaeal clones were observed even in the absence of the
inducer, arabinose. (C) Purified MJ0408 derived protein was incubated with H Neo as described in the methods section. After incubation, the
2
sample was oxidized with iodine to convert the dihydropterins to the fluorescent pterins and assayed by HPLC with fluorescence detection. The first
peak to elute at ∼10 min was neopterin, and the second at ∼16.5 min was 6-hydroxymethylpterin. The figure shows an assay where about half of the
substrate was converted into product. No product was observed at zero time or in an assay run without added enzyme. The MonoQ fraction of the
purified enzyme produced from an E. coli extract not expressing the MJ0408 derived protein likewise did not show any activity. (D) Purified PF0930
derived protein was incubated with 6-HMD and ATP as described in the methods section. After incubation, the sample was oxidized with iodine to
convert the dihydropterins to the fluorescent pterins and assayed by HPLC with fluorescence detection. The first peak to elute at ∼5.2 min was 6-
hydroxymethylpterin-PP, the second at ∼6.6 min was 6-hydroxymethylpterin-P, and the third at 16.5 min was 6-hydroxymethylpterin. The figure
shows an assay where about 90% of the substrate was converted into product. The origin of the 6-hydroxymethylpterin-P is not clear but could arise
from the hydrolysis of the 6-hydroxymethylpterin-PP during sample preparation. No product was observed at zero time or in an assay run without
added enzyme. The MonoQ fraction of the purified enzyme produced from an E. coli extract not expressing the PF0930 derived protein likewise did
not show any activity. Similar results were obtained with the MJ1634 protein.
We then observed that, in Desulfurococcus kamchatkensis, a
gene in the COG2098 family was in a predicted operon with
both the folE2 and COG1634 genes (Figure 3A). Physical
clustering in only one organism is not very strong evidence;
nonetheless, further structural analysis suggested that
COG2098 was the missing archaeal DHNA family. The
COG2098 family previously was targeted by Structural
Genomics Initiatives resulting in the determination of three
representative structures: one from Picrophilus torridus
order. Moreover, the COG2098 subunits assemble in a
compact homotetramer, unlike the tunnel-like architectures of
the canonical DHNA octomer. The tetramer is an apparent
biological unit of the COG2098 family. The most conserved
residues are scattered across the subunit surface but come
together in the subunit interfaces. There are four equivalent
putative active sites in the tetramer, each formed by the
residues from three different subunits (Figure 3B). Fortuitously,
in one of the determined structures, 2OHG, there is a ligand
bound to each of the four sites that was tentatively identified as
8-oxoguanine (8-oxoG). Using the bound ligand as a guide, we
manually docked the predicted substrate molecule in the
COG2098 active site (Figure 3C). The dihydroneopterin
molecule is in essentially the same conformation as in the
structures of canonical DHNA complexes and fits almost
(
PTO0218; PDB: 2I52), one from M. jannaschii (MJ0408;
PDB: 2OGF), and one from Methanopyrus kandleri (MK0786;
PDB: 2IEC). The subunit fold comprising two α-helices and a
four-stranded β-sheet somewhat resembles the fold of bacterial
DHNA in architecture but differs from it in topology, as its
secondary structure elements are connected in a very different
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perfectly in the active site pocket when its pterin ring is aligned
with the 8-oxoG mimic. The environment of bound substrate is
also similar to that of the canonical FolB, suggesting a similar
from the many salts in the sample. Thus, a reaction mixture was
applied to a DEAE-Sephadex HCO3− column (2 × 5 mm), and
the column was washed with 0.5 mL of 0.1 M NH HCO and
4
3
43
enzymatic mechanism for the predicted archaeal DHNA.
the 6-hydroxymethylpterin-PP eluted with 0.5 mL of 0.4 M
NH HCO . After evaporation of the NH HCO , LC-ESI-MS
A genetic approach was first used to validate these
predictions. The COG2098 and the COG1634 encoding
genes from M. jannaschii and represented, respectively, by
4
3
4
3
+
analysis of this sample showed the expected MH ion at 354.1
m/z and (M − H) ion at 352.1 m/z for 6-hydroxymethylpter-
−
mj0408 and mj1634, were cloned into pBAD24 under the P
in-PP. The identity of the compound was further confirmed by
the measurement of the MRM 354/125 and 354/176
fragments, the same as observed for the known sample of 6-
hydroxymethylpterin-PP.
BAD
4
4
promoter and tested for complementation of the dT
R
auxotrophy phenotypes of the ΔfolB::Kan E. coli strain
3
1
R
(
VDC3267 ) and of the ΔfolK::Tet E. coli strain (C600
4
5
ΔfolK::tetB ), respectively. E. coli strains deleted in H -folate
4
The combination of genetic and biochemical data presented
here strongly validates our comparative genomic derived
predictions solving the long-standing mystery of 6-HMDP
biosynthesis in most Archaea. We therefore renamed the two
families MptD for the archaeal specific DHNA and MptE for
the archaeal specific 6-HMDPK. The discovery of the missing
archaeal 6-HMDP synthesis genes completes the picture for the
initial steps in the pterin pathways in the third kingdom of life
biosynthesis genes can grow on rich medium if dT is added to
the medium, albeit poorly because of the absence of
46
formylation of the initiator tRNA. As shown on Figure 4A,
expression of mj0408 complemented the dT auxotrophy
phenotype of the folB deletion as did expression of the E. coli
folB positive control. Overexpression of mj0408 seemed to be
toxic with cells showing better growth with no arabinose
(
Figure 4A). Similarly, overexpression of mj1634 comple-
(
Figures 1 and 2). A great diversity in the metabolic and
mented the dT auxotrophy phenotype of the folK deletion as
did expression of the E. coli folK positive control (Figure 4B).
A biochemical validation strategy was then used to confirm
the genetic results. mj0408, mj1634, and its homologue from P.
furiosis (pf0930) were all expressed in E. coli. The respective
gene products, MJ0408, MJ1634, and PF0930 were purified by
heating the extract to 80 °C followed by anion exchange
chromatography. The resulting proteins were greater than 95%
pure as judged by polyacrylamide gel electrophoresis with
coomassie staining (Supplemental Figure 4). The identity of
the purified proteins was confirmed by MALDI MS of the
tryptic-digested protein band. The protein product from
mj0408 was confirmed to be a DHNA as it was found to
enzymatic solutions used to produce this molecule is observed,
and the picture may be even more complex as some genes are
still missing in specific lineages. MtpE homologues are found in
almost all archaeal genomes (Figure 2). The handful of
organisms that lack this gene like Nanoarchaeum equitans, or
Staphylothermus marinus have lost all other 6-HMDP biosyn-
47
thesis genes and must certainly salvage pterin cofactors, even
if one cannot rule out missing genes without further studies.
For MptD, the situation is more complex. As discussed above, if
the majority of archaeal genomes analyzed encode an MptD
homologue, a minor subset does not. It seems most
Thermococcales use a PTPS-III dependent bypass (Figures 1
and 2 and Supplemental Information), whereas Sulfolobus
catalyze the formation of 6-HMD from H Neo (Figures 1 and
2
31
solfataricus uses a PTPS-VI dependent one. A few archaeal
4C). The retention time of the 6-hydroxymethylpterin was
species known to synthesize pterin containing C1-carrier
identical to that of the 6-hydroxymethylpterin standard under
two separate chromatographic systems utilizing either a Varian
PursuitXRs C18 column or a Varian Pursuit polyfluorophenyl
48
coenzymes such as Archaeoglobus fulgidus or Pyrobaculum
10
species lack homologues of MptD, FolB, PTPS-III, or PTPS-
VI. A few of these (mainly Thermoproteales) encode members
of the PTPS-V family (Figure 2), but initial validation tests with
the Pyrobaculum calidifontis PTPS-V encoding gene, Pcal_1063,
(
PFP) column. The formation of 6-hydroxymethylpterin was
linear with respect enzyme concentration, and no product was
observed in control samples that were incubated in the absence
of enzyme. In order to confirm that the observed activity was
due to the mj0408 gene product and not a result of the E. coli
DHNA activity, the activity of a cell extract and identical
purified fractions from E. coli expressing the mj0408 gene to
those of E. coli expressing a different gene (mj0929) were
compared. The DHNA activity was greater than 4-fold higher
in cell extracts of E. coli expressing the mj0408 gene. The
identical MonoQ-purified fraction of mj0929 exhibited no
activity relative to the purified mj0408 gene product.
The protein product from pf0930 was confirmed to be a 6-
HMDPK as it was found to catalyze the formation of 6-HMDP
from 6-HMD and ATP (Figures 1 and 4D). Of the four
nucleotide phosphates tested, the maximum activity was
observed with ATP. Relative to ATP, CTP, UTP, and GTP
had, respectively, 41%, 40%, and 12% of the ATP activity. The
fact that the oxidized product peak was 6-hydroxymethylpterin
diphosphate (6-hydroxymethylpterin-PP) was confirmed by the
following observations. The product peak had the same UV/
visible absorbance and fluorescence spectra as a known sample
of 6-hydroxymethylpterin-PP. Attempts to confirm the identity
of the product peak by LC-ESI-MS analysis of crude incubation
mixtures was not successful most likely due to ion suppression
31
were negative. Some archaeal genomes such as M. barkeri
encode both MptD and PTPS-III homologues and others such
as S. solfataricus encode both FolB and PTPS-VI homologues
(
Figure 2). Of note, these organisms synthesize both H -folate
4
1
3,15,49
and H -MPT derivatives or hybrid molecules.
There are
4
differences between the uses H -folate and H -MPT as C1
4
4
7
10
donors since H -MPT derivatives do not form N -formyl
4
derivatives as a result of thermodynamic differences in the
chemical properties of the arylamine nitrogen N1 in H -folate
0
4
and H -MPT. In organisms that use both cofactors, it might be
4
necessary to use two different gene families in order to
independently control the production of these molecules.
The discovery of MptD and MptE provides new examples of
50
both divergent and convergent enzyme evolution. Further
characterization of these enzymes therefore will be of interest
for structural biologists and biochemists. Finally, folate
biosynthesis genes are traditional antibacterial targets and
recently methanopterin biosynthesis has been proposed as a
target to eliminate the dominant archaeon in the human gut
51
Methanobrevibacter smithii. Both MptD and MptE represent
new targets found neither in human nor other members of the
52
bacterial flora. As new roles of the gut Euryarchaeota emerge,
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inhibiting methanopterin pathway enzymes might be a viable
solution to selectively eliminate Archaea from the flora.
harvested by centrifugation (4000 × g, 5 min) and frozen at −20 °C.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of total cellular
proteins confirmed induction of the desired protein at approximately
1
4 kDa for MJ0408 and 27.6 kDa for MJ1634.
Purification of Recombinant MJ0408 and MJ1634 Gene
METHODS
■
Products. Frozen E. coli cell pellets (∼0.4 g wet weight from 200 mL
of medium) were suspended in 3 mL of extraction buffer (50 mM N-
Bioinformatics. Analysis of the phylogenetic distribution and
physical clustering was performed in the SEED database on the 58
genomes available at the time of the analysis (Dec/2011). Results are
available in the “Pterin Biosynthesis Archaea” subsystem on the public
SEED server (http://pubseed.theseed.org/SubsysEditor.cgi). A subset
of the analysis is summarized Figure 2. We also used the BLAST tools
2
[
7
tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), pH
.0, 10 mM MgCl , 20 mM DTT) and lysed by sonication. Both
2
protein products were found to remain soluble after heating the
resulting cell extracts for 10 min at 70 °C followed by centrifugation
5
3
(16 000 × g for 10 min). This process allowed for the purification of
and resources at NCBI5. Multiple sequence alignments were built
4
the desired enzymes from the majority of E. coli proteins, which
denature and precipitate under these conditions. The next step of
purification was performed by anion-exchange chromatography of the
using the ClustalW tool. Structure based alignments were performed
using the Espript platform (http://espript.ibcp.fr/ESPript/ESPript/
55
)
.
Visualization and comparison of protein structures and manual
docking of ligand molecules were performed using PyMol (The
PyMOL Molecular Graphics System, Version 1.4.1, Schrodinger,
LLC).
Chemicals. 7,8-Dihydroneopterin, 6-hydroxymethylpterin-mono-
phosphate (6-hydroxymethylpterin-P), 6-hydroxymethylpterin-PP, 6-
hydroxymethyl-7,8-dihydropterin, 6-hydroxymethyl-7,8-dihydropterin-
P, 6-hydroxymethyl-7,8-dihydropterin-PP, and D-neopterin were
supplied by Schircks Laboratories, Jona, Switzerland. ATP, GTP,
CTP, UTP, and 6-hydroxymethylpterin was supplied by Sigma.
Recombinant NgFolE2 was supplied by Dirk Iwata-Reuyl, Department
of Chemistry, Portland State University, Portland, Oregon.
7
0 °C soluble fractions on a MonoQ HR column (1 × 8 cm;
Amersham Bioscience) using a linear gradient from 0 to 1 M NaCl in
5 mM Tris buffer (pH 7.5), over 55 min at a flow rate of 1 mL/min.
̈
2
Fractions of 1 mL were collected. The different purification steps are
shown in Supplemental Figure 4 for MJ1634 as prototypical of the
purification of all the proteins described herein. Protein concentrations
were determined by Bradford analysis.
Cloning of the P. furiosus pf 0930 and pf1278 Genes and
Expression of Their Gene Products. The recombinant plasmids
pPF0930 and pPF1278 were constructed as described in Sugar et al.
5
8
Briefly, the primers used for pf 0930 were PF0930For (5′-
CACCGGATCCAAGTGGGAGGAGTGGAAGCCATTC-3′) and
PF0930Rev (5′-AAGCTCGAGCGGCCGCGATTTACGACTTGA-
GATAATAAAAAC-3′). The primers used for pf1278 were
PF1278For (5′-CACCGGATCCAAGGCTAGGATTATCTATA-
GAGCT-3′) and PF1278Rev (5′-AAGCTCGAGCGGCCG-
CAGTGGTAAGGTCAAGGTGAGGTTTGA-3′). These primers
were used to amplify the genes by PCR and cloned into a modified
pET24d vector using BamHI and NotI restriction enzymes. Proteins
E. coli Complementation Tests. The mj0408 (NP_247382.1)
and mj1634 (NP_248644.1) corresponding genes were amplified from
M. jannaschii genomic DNA by PCR and cloned into the NcoI and
R
44
SphI sites of pBAD24 (Amp , ColE1) after digestion with the
corresponding enzymes to give plasmids pGPP528 (or pMJ0408) and
pGPP541 (or pMJ1634), respectively. The resulting plasmids were
verified by Sanger sequencing at the University of Florida core facility.
Primers used were MjfolB2_Fwd (5′-CGTGACCATGGGAGTA-
GAAGAAACAGAAG-3′) and MjfolB2_Rev (5′-GGTCGGCATGCT-
TATTCCTCAAACTTTTTGACATAC-3′) for the mj0408 cloning
and MJ1634pBAD24_Fwdol1 (5′-ATCGGCCATGGACATGAAG-
GAGTGGGA-3′) and MJ1634pBAD24_Revol2 (5′-
GGTCGGCATGCTTATTTTAAAAATTCAATCTCTATTT-3′) for
the mj1634 cloning. Positive controls were used as plasmids expressing
the WT E. coli folB (pBAD24::folB , Amp , ColE1) and the WT E.
coli folK gene (pfolK , ASKA clone JW0138 ). E. coli derivatives were
routinely grown at 37 °C in LB (BD Diagnostic System). Growth
media were solidified with 15 g/L agar (BD Diagnostic System) for
the preparation of plates. Transformations of E. coli were performed
following standard procedures. Ampicillin (Amp, 100 μg/mL),
thymidine (dT, 80 μg/mL), chloramphenicol (20 μg/mL), kanamycin
58
encoded by these plasmids were expressed and purified as described
Enzymatic Assay of DHNA Activity. The standard assay used for
the measurement of DHNA enzymatic activity was conducted in 200
μL reaction volume and included 7 ng of M. jannaschii MJ0408, 40
mM TES/KCl buffer pH 7.4, 8 mM MgCl , 16 mM DTT, and 110 μM
H Neo. Samples were sealed under argon and incubated for 10 min at
70 °C. Following incubation, the reactions were quenched by the
addition of 20 μL of 1 M HCl. H Neo and 6-HMD in the incubation
mixture were oxidized to fluorescent neopterin and 6-hydroxyme-
thylpterin by the addition of 8 μL of a saturated solution of iodine in
methanol and incubated at RT for 30 min. Following oxidation, the
samples were neutralized by the addition of 20 μL of 1 M NaOH and
excess iodine removed by reduction with 8 μL of 1 M NaHSO3.
Following centrifugation, the samples were combined with water for a
final volume of 1 mL and analyzed by HPLC as described below.
Enzymatic Assay of 6-HMDPK Activity. The standard assay used
for the measurement of 6-HMDPK enzymatic activity was conducted
in 200 μL reaction volume and included 3.5 ng of the PF0930 (or
2
2
R
31
Ec
56
2
Ec
57
(
Kan, 50 μg/mL), and L-arabinose (0.02% to 0.2%) were used as
needed.
Cloning and expression of the M. jannaschii mj0408 and
mj1634 and expression of their gene products. The M.
jannaschii genes mj0408 and mj1634 was amplified from M. jannaschii
genomic DNA by PCR. The primers used for mj0408 were
mj0408Fwd (5′-GGTCATATGAGAGTAGAAGAAACAGAAG-3′)
and MJ0408Rev (5′-GCTGGATCCTTATTCCTCAAACTTTTT-
GAC-3′). The primers used for mj1634 were mj1634Fwd (5′-
GGTCATATGGACA-TGAAGGAGTG-3′) and mj1634Rev (5′-
GCTGGATCCTTATTTTAAAAATTCAATCTC-3′). PCR amplifi-
cation was performed using a 55 °C annealing temperature for mj0408
and 50 °C for mj1634. The PCR product was purified, digested with
NdeI and BamHI restriction enzymes, and then ligated into compatible
sites in plasmid pT7−7 to make the recombinant plasmid pMJ0408
and pMJ1634. The sequences were verified by sequencing at the
University of Iowa DNA core facility. The resulting plasmids were
transformed into E. coli strain BL21-Codon Plus (DE3)-RIL
MJ1634) enzyme, 40 mM TES/KCl buffer pH 7.4, 8 mM MgCl , 16
2
mM DTT, 100 μM 6-HMD, and 1 mM ATP. Samples were sealed
under argon and incubated for 10 min at 70 °C. Following incubation,
the reactions were quenched by the addition of 20 μL of 1 M HCl. 6-
HMD and 6-HMDP in the incubation mixture were oxidized to the
fluorescent pterins by the addition of 8 μL of a saturated solution of
iodine in methanol and incubated at RT for 30 min. Following
oxidation, the samples were neutralized by the addition of 20 μL 1 M
NaOH and excess iodine removed by reduction with 8 μL of 1 M
NaHSO . Following centrifugation, the samples were combined with
water for a final volume of 1 mL and analyzed by HPLC as described
below.
HPLC Analysis of Pterins. Chromatographic separation of pterins
was performed on a Shimadzu HPLC System with a C18 reverse phase
column (Varian PursuitXRs 250 × 4.6 mm, 5 μm partical size). The
elution profile consisted of 5 min at 95% sodium acetate buffer (25
3
(
Stratagene). Transformed cells were grown in LB-medium (200
mL) supplemented with 100 μg/mL ampicillin at 37 °C with shaking
until they reached an OD600 of 1.0. Recombinant protein production
was induced by the addition of lactose to a final concentration of 28
mM. After an additional 4 h of culture at 37 °C, the cells were
mM, pH 6.0, 0.02% NaN ) and 5% MeOH followed by a linear
gradient to 20% sodium acetate buffer/80% MeOH over 40 min at 0.5
3
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Letters
mL/min. Pterins were detected by fluorescence using an excitation
wavelength of 356 nm and an emission wavelength of 450 nm. Under
these conditions, pterins were eluted in the following order (min): 6-
hydroxymethylpterin-PPP (4.982), 6-hydroxymethylpterin-PP (5.243),
(6) Grochowski, L. L.; White, R. H. (2010) Biosynthesis of the
Methanogenic coenzymes. In Comprehensive Natural Products II:
Chemistry and Biology (Begley, T., Ed.), pp 711−748, Elsevier, New
York.
6
(
-hydroxymethylpterin-P (6.605), D-neopterin (10.10), monapterin
12.012), and 6-hydroxymethylpterin (16.490). Alternately, pterins
were separated on a Varian Pursuit polyfluorophenyl (PFP) column
250 × 4.6 mm, 5 μm partical size). The elution profile was isocratic
(7) Maden, B. E. (2000) Tetrahydrofolate and tetrahydrometha-
nopterin compared: functionally distinct carriers in C1 metabolism.
Biochem. J. 350, 609−629.
(
(8) Chistoserdova, L., Vorholt, J. A., Thauer, R. K., and Lidstrom, M.
with 95% formic acid in water (0.1%) and 5% MeOH. Pterins were
detected by fluorescence using an excitation wavelength of 356 nm and
an emission wavelength of 450 nm.
E. (1998) C1 transfer enzymes and coenzymes linking methylotrophic
bacteria and methanogenic Archaea. Science 281, 99−102.
(9) Ortenberg, R., Rozenblatt-Rosen, O., and Mevarech, M. (2000)
The extremely halophilic archaeon Haloferax volcanii has two very
different dihydrofolate reductases. Mol. Microbiol. 35, 1493−1505.
ASSOCIATED CONTENT
■
(
10) White, R. H. (1991) Distribution of folates and modified folates
*
S
Supporting Information
Ezymatic and HPLC assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
in extremely thermophilic bacteria. J. Bacteriol. 173, 1987−1991.
(11) White, R. H. (1993) Structures of the modified folates in the
extremely thermophilic archaebacterium Thermococcus litoralis. J.
Bacteriol. 175, 3661−3663.
AUTHOR INFORMATION
■
*
(12) White, R. H. (1993) Structures of the modified folates in the
thermophilic archaebacteria Pyrococcus furiosus. Biochemistry 32, 745−
Corresponding Author
Tel: (352) 392 9416. Fax: (352) 392 5922. E-mail: vcrecy@
ufl.edu.
7
53.
(
13) White, R. H. (1997) Purine biosynthesis in the domain Archaea
without folates or modified folates. J. Bacteriol. 179, 3374−3377.
14) Thauer, R. (1997) Biodiversity and unity in biochemistry.
Antonie van Leeuwenhoek 71, 21−32.
15) Buchenau, B., and Thauer, R. K. (2004) Tetrahydrofolate-
Notes
(
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
specific enzymes in Methanosarcina barkeri; and growth dependence of
this methanogenic archaeon on folic acid or p-aminobenzoic acid.
Arch. Microbiol. 182, 313−325.
■
We thank G. Swedberg for the E. coli ΔfolK strain. We thank M.
Rasche and A. Hanson for frequent discussions and input and
critical reading of the manuscript. We thank C. Brochier-
Armanet for providing an archaeal genetic tree template. We
thank D. Miller with helping to purify the M. jannaschii
enzymes and D. Iwata-Reuyl for providing purified FolE2 from
Neisseria gonorrhoeae. This research was supported in part by
the U.K. Medical Research Council U1051922716 to A.G.M.,
the U.S. National Science Foundation MCB 0722787 to
R.H.W., and the National Institutes of Health GM070641 to
V.d.C.-L.
(16) Zhou, D., and White, R. H. (1992) 5-(p-Aminophenyl)-1,2,3,4-
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Sulfolobus solfataricus. J. Bacteriol. 174, 4576−4582.
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nomics. Nat. Prod. Rep. 19, 133−147.
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NOTE ADDED AFTER ASAP PUBLICATION
This paper was published ASAP on September 7, 2012. Figure
legend has been updated. The revised version was posted on
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