Purine biosynthesis in archaea: variations on a theme - Biology Direct

21 Pages·2012·1.64 MB·English

by  Anne M Brown

Brownetal.BiologyDirect2011,6:63
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RESEARCH Open Access
Purine biosynthesis in archaea: variations on a
theme
Anne M Brown1,2, Samantha L Hoopes1,3, Robert H White2 and Catherine A Sarisky1*
Abstract
Background: The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of
life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and
bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates
the value of manual curation in archaea, even in pathways that have been well-studied in other domains.
Results: We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11
steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence
similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active
site residues and other residues known or suspected to be required for function.
Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide
ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two
more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of
formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide
ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.
Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins
without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of
proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%)
assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a
specific enzyme name and E. C. number were well-justified.
Conclusions: The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has
been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can
improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a
need to identify further pathway components even in well-studied pathways.
Reviewers: This article was reviewed by Dr. Céline Brochier-Armanet, Dr Kira S Makarova (nominated by Dr. Eugene
Koonin), and Dr. Michael Galperin.
Background study of archaea and the availability of archaeal gen-
Purines are key components of all living cells on earth, omes, it became clear that the purine biosynthesis path-
required for energy metabolism and biosynthesis of way in many archaea included several unique enzymes
RNA and DNA. Purine biosynthesis pathways were first [7,8].
described in the 1950’s and 1960’s [1-3] and represented The accepted purine biosynthesis pathway (with
a central force in the development of the field of bio- known variations) is shown in Figure 1. There is com-
chemistry. For decades, the story of purine biosynthesis plete conservation of the intermediates of purine bio-
seemed mostly complete, with only a few new enzymes synthesis from phosphoribosyl pyrophosphate (PRPP) to
added to the pathway [4-6]. However, with increased 5-phospho-b-D-ribosylamine (PRA), with the exception
of N5-CAIR (N5-carboxyaminoimidazole ribonucleotide),
*Correspondence:[email protected] which is bypassed in eukaryotes. The enzymes catalyzing
1DepartmentofChemistry,RoanokeCollege,Salem,VA24153,USA
each step, however, are more variable, with four
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Figure1ThedenovobiosynthesisofIMP.Forclarity,cosubstratesarenotshown.Abbreviationsareasusedinthetext.
common nonhomologous enzyme substitutions known final two steps of the de novo purine pathway present
across the three domains. Intriguingly, the archaea man- whenever the histidine biosynthesis pathway is present.
ifest all four of the known nonhomologous substitutions The availability of full genome sequence data for
in this pathway, with evidence (discussed in this paper) numerous archaeal species allows the comparative study
for an additional two substitutions still to be identified. of the enzymes involved in de novo purine biosynthesis.
The de novo purine biosynthesis pathway from PRPP We used the Integrated Microbial Genome system
to IMP includes one branch, to thiamine biosynthesis, (IMG) to curate open reading frames (ORFs) on the
and one alternate input, from histidine biosynthesis. basis of high similarity to an experimentally character-
Thiamine biosynthesis uses AIR (aminoimidazole ribo- ized gene or enzyme in another species (ideally as a
tide) as a substrate. Thus, the first four enzymatic steps bidirectional best hit [9]). The presence of nearby gene
of the pathway are not truly committed to purine bio- candidates for the same biochemical pathway supported
synthesis, but serve both purine biosynthesis and thia- the annotations through “guilt by association” [10].
mine biosynthesis. The interaction between the histidine Although several metabolic pathway reconstructions are
pathway and purine biosynthesis is less easily dia- available elsewhere [11,12], a genomic analysis of the
grammed. ATP (an end-product of purine biosynthesis purine biosynthesis pathway of the archaea in the depth
or obtained by salvage) reacts with PRPP, followed by a and breadth presented here is not otherwise available.
ring-opening reaction that opens the purine ring, lead- Below, we describe our findings upon inspection of each
ing to production of aminoimidazole carboxamide ribo- genome and discuss these findings in the context of pre-
nucleotide (AICAR), which can be formylated and then vious experimental data.
cyclized to re-form the purine ring. Thus, provided that The ever-increasing pace of genome sequencing
the final two steps of the de novo purine biosynthesis means that genomes will increasingly be annotated by
pathway are functional, histidine biosynthesis will result automated or semi-automated processes. This paper
in no net loss or gain in purines. Due to the desirability serves as a case study of the errors possible in the lar-
of reclaiming the AICAR, we expect to find at least the gely automated gene annotations present in IMG. As
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purine biosynthesis is relatively well-understood, our eleven steps in the pathway. Redundancy was especially
findings represent a best-case scenario, with increased common in the AICAR formyltransferase assignments.
likelihood of problem annotations in pathways that have Only in the case of the purB and purM genes could we
been less well-studied. assign exactly one gene from each purine biosynthesiz-
ing organism to the necessary function. Specific findings
Results and Discussion for each biosynthetic step are discussed below.
We identified a complete or nearly complete set of the
genes necessary for purine biosynthesis in 58 of the 65 Organisms that do not synthesize purines
species studied. However, 25 of these suspected purine Our analysis revealed seven archaea with entirely absent
biosynthesizing species lacked a gene candidate for or almost entirely absent genes encoding purine bio-
either glycinamide ribonucleotide (GAR) formyltransfer- synthesis enzymes. On the basis of the genomic data,
ase or inosine 5’-monophosphate (IMP) cyclohydrolase. these organisms are incapable of biosynthesis of purines
As each of these 25 species lacked just one gene in the and must rely on environmental sources coupled with
pathway, we believe that these species do synthesize salvage pathways to meet their purine requirements.
purines, and that further work will reveal novel pathway The non-purine synthesizing organisms identified are:
variants. The gene candidates for each of the purine bio- Aeropyrum pernix K1, Korarchaeum cryptofilum OPF8,
synthetic steps are listed in Additional File 1, with a Hyperthermus butylicus DSM 5456, Nanoarchaeum
summary of findings in Figures 2 and 3. equitans Kin4-M, Thermococcus onnurineus NA1, Sta-
We identified at least one organism with apparent phylothermus marinus F1, and Thermofilum pendens
redundancy (either two similar copies of the same gene Hrk 5. Based on the distribution of these organisms
or two different genes for the same step) in five out of throughout the archaeal domain, the loss of purine
Purine biosynthesis proteins
Genome Name F D N T U S L Q M K E C B H1 P H2 O
Crenarchaeota
Thermofilum pendens Hrk 5
Thermoproteus neutrophilus V24Sta ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Caldivirga maquilingensis IC-167 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Pyrobaculum aerophilum IM2 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ? Thermoproteales
Pyrobaculum islandicum DSM 4184 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Pyrobaculum calidifontis JCM 11548 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Pyrobaculum arsenaticum DSM 13514 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Staphylothermus marinus F1 □
Ignicoccus hospitalis KIN4/I ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Desulfurococcus kamchatkensis 1221n ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ? Desulfurococcales
Aeropyrum pernix K1
Hyperthermus butylicus DSM 5456
Metallosphaera sedula DSM 5348 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Sulfolobus tokodaii 7 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Sulfolobus islandicus L.S.2.15 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ? Sulfolobales
Sulfolobus solfataricus P2 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Sulfolobus acidocaldarius DSM 639 ■,□ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?

Korarchaeota
Korarchaeum cryptofilum OPF8 ■ ■,▲

Nanoarchaeota
Nanoarchaeum equitans Kin4-M

Thaumarchaeota
Cenarchaeum symbiosum A ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Nitrosopumilus maritimus SCM1 ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■,▲ ?
Figure 2 The presence of genes for the purine biosynthesis pathway in Crenarchaeota, Nanoarchaea, Thaumarchaeota, and
Korarchaea.Aschematic,taxonomy-basedphylogenetictreeisprovided,alongwithordernameswithintheCrenarchaota.Thetypicallybi-
functionalPurHproteinappearstwice,asPurH1(theC-terminalAICARformyltransferasedomain)andPurH2(theN-terminalIMPcyclohydrolase
domain).AdditionalFile1containsgenelocustagsforeachcandidategene.Symbolsused:■denotesagenethatisagoodmatch.□denotes
amatchwithsomeproblems,asdescribedinmoredetailinthetext.▲isusedtorepresentaclusterIIPurPprotein.[■]indicatesthatthe
expectedgeneissplitintotwoadjacentloci.■-■denotesaproteinwithadomainduplication.Wherea“?”appears,ageneisnecessaryforan
otherwisecompletepurinebiosynthesispathwaytobefunctional,butnogenecandidatecouldbeidentifiedwiththedataavailable.
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(cid:87)(cid:455)(cid:396)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:296)(cid:437)(cid:396)(cid:349)(cid:381)(cid:400)(cid:437)(cid:400)(cid:3)(cid:24)(cid:94)(cid:68)(cid:3)(cid:1007)(cid:1010)(cid:1007)(cid:1012)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:376)(cid:3) (cid:845)(cid:3)
(cid:87)(cid:455)(cid:396)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:258)(cid:271)(cid:455)(cid:400)(cid:400)(cid:349)(cid:3)(cid:39)(cid:28)(cid:1009)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:376)(cid:3) (cid:845)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:364)(cid:381)(cid:282)(cid:258)(cid:364)(cid:258)(cid:396)(cid:258)(cid:286)(cid:374)(cid:400)(cid:349)(cid:400)(cid:3)(cid:60)(cid:75)(cid:24)(cid:1005)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:401)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:401)(cid:853)(cid:376)(cid:3) (cid:374)(cid:3) (cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:258)(cid:367)(cid:286)(cid:400)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:336)(cid:258)(cid:373)(cid:373)(cid:258)(cid:410)(cid:381)(cid:367)(cid:286)(cid:396)(cid:258)(cid:374)(cid:400)(cid:3)(cid:28)(cid:58)(cid:1007)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:401)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:896)(cid:401)(cid:897)(cid:853)(cid:376)(cid:3) (cid:374)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:381)(cid:374)(cid:374)(cid:437)(cid:396)(cid:349)(cid:374)(cid:286)(cid:437)(cid:400)(cid:3)(cid:69)(cid:4)(cid:1005)(cid:3)(cid:3) (cid:374)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:272)(cid:381)(cid:272)(cid:272)(cid:437)(cid:400)(cid:3)(cid:400)(cid:349)(cid:271)(cid:349)(cid:396)(cid:349)(cid:272)(cid:437)(cid:400)(cid:3)(cid:68)(cid:68)(cid:3)(cid:1011)(cid:1007)(cid:1013)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:853)(cid:376)(cid:3) (cid:845)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:393)(cid:367)(cid:258)(cid:400)(cid:373)(cid:258)(cid:3)(cid:448)(cid:381)(cid:367)(cid:272)(cid:258)(cid:374)(cid:349)(cid:437)(cid:373)(cid:3)(cid:39)(cid:94)(cid:94)(cid:1005)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3)
(cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:393)(cid:367)(cid:258)(cid:400)(cid:373)(cid:258)(cid:3)(cid:258)(cid:272)(cid:349)(cid:282)(cid:381)(cid:393)(cid:346)(cid:349)(cid:367)(cid:437)(cid:373)(cid:3)(cid:24)(cid:94)(cid:68)(cid:3)(cid:1005)(cid:1011)(cid:1006)(cid:1012)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:100)(cid:346)(cid:286)(cid:396)(cid:373)(cid:381)(cid:393)(cid:367)(cid:258)(cid:400)(cid:373)(cid:258)(cid:410)(cid:258)(cid:367)(cid:286)(cid:400)(cid:3)
(cid:87)(cid:349)(cid:272)(cid:396)(cid:381)(cid:393)(cid:346)(cid:349)(cid:367)(cid:437)(cid:400)(cid:3)(cid:410)(cid:381)(cid:396)(cid:396)(cid:349)(cid:282)(cid:437)(cid:400)(cid:3)(cid:24)(cid:94)(cid:68)(cid:3)(cid:1013)(cid:1011)(cid:1013)(cid:1004)(cid:3)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3) (cid:374)(cid:3)
Figure3ThepresenceofgenesforthepurinebiosynthesispathwayinEuryarchaeota.SymbolsareasinFigure2.Eindicatesthatthe
expectedactivitycanbeprovidedbythePurEprotein(seetext).LindicatesthattheexpectedproteinsequenceisencodedaspartofthePurL
protein(seetext).
biosynthesis genes has occurred multiple times. These (respectively), either of which would provide a purine
organisms entirely lack the genes required for de novo source. Similarly, Thermofilum pendens, believed to be
purine biosynthesis enzymes, with only minor excep- adapted to growth in nutrient-rich environments,
tions, described below. requires tryptone or yeast extract for culture [17]. The
Where available, the growth conditions of these non- parasitic Nanoarchaeum equitans must rely on Ignicoc-
purine biosynthesizing organisms are consistent with a cus hospitalis to supply its purine requirement [18].
requirement for purines. Aeropyrum pernix requires In T. onnurineus, which grows in rich media that
adenine in its growth medium, consistent with an inabil- could meet the organism’s apparent purine requirement
ity to synthesize purines de novo [13]. Korarchaeum [19], only a purB-like gene is present, probably involved
cryptofilum has not been grown in pure culture [14]. in purine interconversion. (We also found the purine
Hyperthermus butylicus [15] and Staphylothermus mari- interconversion genes guaA, guaB, and purA, not
nus [16] (both sulfur-reducing peptide fermenters) grow shown.) Although Lee and coworkers have proposed
with tryptone or peptone in the growth medium that T. onnurineus obtains purines through its histidine
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biosynthesis pathway [19], de novo histidine biosynthesis N-terminus [21]. In E. coli, the cysteine is N-terminal
provides no net creation of purines due to use of the after removal of the initial methionine. In E. coli, the N-
purine ring of ATP as a substrate, as described above. terminal cysteine is required for use of glutamine as the
Additionally, there is no genomic evidence of a PurP or nitrogen source, with no detectable activity when the
PurH enzyme that could convert the aminoimidazole cysteine is replaced with alanine or serine, although
carboxamide ribonucleotide (AICAR) produced by histi- ammonia can still serve as the nitrogen source [22]. We
dine biosynthesis back into a purine. If T. onnurineus thus expect any archaeal purF gene should encode a
truly lacks a means to convert the AICAR produced by cysteine at or near the N-terminus if glutamine is the
histidine biosynthesis back to a purine, biosynthesis of nitrogen source.
histidine will in fact result in a net loss of purines and We identified a gene encoding a protein similar to
accumulation of AICAR in this organism. Thus, we sus- PurF in all of the purine biosynthesizing archaeal species
pect that T. onnurineus is auxotrophic for purines, with studied, as shown in Figures 2 and 3. Out of the Eur-
higher need for purines (and accumulation of AICAR) yarchaeota studied, only Methanocorpusculum labrea-
in the absence of environmental histidine. num’s genome includes two candidate sequences,
In the case of K. cryptofilum, which has been enriched Mlab_0202 and Mlab_0150. As Mlab_0150’s predicted
only in complex media (in which purines would be pre- protein has a serine in place of the active site cysteine,
sent) and is thought to use peptides as an energy source we suspect that Mlab_0150 does not encode a glutamine
[14], we identified a pair of purP-like genes, along with phosphoribosylpyrophosphate amidotransferase, and that
purB. As PurB functions in purine interconversion as Mlab_0202 is the actual purF gene. Mlab_150 appears to
well as de novo biosynthesis, we do not consider its pre- be a relatively recent duplication of Mlab_0202, as it is
sence indicative of de novo purine synthesis. The role of most similar to Mlab_0202 and the putative purFs in
PurP-like enzymes, however, is not entirely clear in this closely related species.
organism, as will be discussed in further detail below. The most promising PurF candidates in Methanosaeta
thermophila, Desulfurococcus kamchatkensis, and Cen-
Glutamine phosphoribosylpyrophosphate archaeum symbiosum initially appeared to be lacking
amidotransferase (EC 2.4.2.14, GPATase, PurF) the active site cysteine, but when we inspected the 5’
The purF gene product, glutamine phosphoribosylpyro- DNA sequence, we found that a cysteine was present if
phosphate amidotransferase (GPATase, PurF), catalyzes we proposed an earlier translation initiation site, as
the first step in biosynthesis of purines, conversion of shown in Figure 4. Some archaeal PurF candidates
phosphoribosyl pyrophosphate (PRPP) to 5-phospho-b- require cleavage of several residues to reveal the N-
D-ribosylamine (PRA). The Escherichia coli PurF terminal cysteine and others require only removal of the
enzyme has also been demonstrated to have low-level N-terminal methionine. Although experimental data is
promiscuity for phosphoribosylanthranilate isomerase required to verify these translation initiation sites, our
activity (normally encoded by trpF), as demonstrated by findings are consistent with at least 5% error rate (3/58)
complementation studies [20]. for initiation site determination in these purF genes.
A conserved N-terminal cysteine residue forms a cata- Alignment and phylogenetic tree generation for the
lytic triad with histidine and aspartate in the glutamine two PurF-like proteins in each of the Crenarchaeota
amidotransfer domain. In Bacillus subtilis, this is revealed that two distinct clusters (Additional Files 1
accomplished by cleavage of a short peptide from the and 2), with each Crenarchaeon having one gene in
Figure4N-terminalsequencesfromselectedPurFcandidates.StartsitesinIMGareunderlined,withproteinsequencesforourproposed
earlierstartsitesshownforDKAM_0523,Mthe_0230,andCENSYa_1910.Thecysteinenucleophileisinbold.
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each cluster. One cluster contained proteins with the proteins were the only PurD-like sequences we identi-
expected active-site N-terminal cysteine, and we believe fied in which a highly conserved aspartate in the R[LF]
these are the actual PurF enzymes. The proteins in the GDPEx[EQIM] motif (Prosite PS00184, corresponding
second cluster, which we call PurF’, lacked the N-term- to amino acids 290-298 in the E. coli PurD) was not
inal cysteine necessary for glutaminase activity. Many conserved [27]. Similarly, the N-terminal P-loop region
PurF’ proteins had alanine or valine, which could not is quite divergent in Hlac_1553 and rrnAC0307 com-
substitute as the nucleophile required for glutaminase pared to the other archaeal PurD-like proteins. We con-
activity. These purF’ gene loci are enclosed in parenth- clude that these “extra” proteins are not actual
eses in Additional File 1 and denoted with “□” in Figures glycinamide ribonucleotide synthetases, despite their
2 and 3 to indicate the missing active site feature. The annotations.
genes purF and purF’ are close to each other and to
other purine biosynthesis genes in these genomes; thus, Formate-dependent GAR formyltransferase (EC 6.3.4.-,
gene clustering supports the role of both PurF and PurT) and GAR formyltransferase (EC 2.1.2.2, PurN)
PurF’ in purine biosynthesis. Many of the Crenarchaeota Conversion of GAR to formyl glycinamide ribonucleo-
have other ORFs that are bidirectional best hits for the tide (FGAR) may be catalyzed by either of two distinct
similar amidotransferases AsnB and GlmS, so we do not formyltransferases. The first, PurN, transfers a formyl
believe that either of these purF-like genes is actually group from 10-formyltetrahydrofolate to GAR. The sec-
asnB or glmS [23]. As both PurF and AsnB exhibit a ond, PurT, uses formate as the one-carbon source for
loss of function when the equivalent cysteine residue is formylation of GAR, with coupled hydrolysis of ATP. In
replaced with alanine or serine [24], the PurF’ proteins those archaea that use only methanopterin-related coen-
are unlikely to be functional amidotransferases with any zymes as C-1 carriers, with no 10-formyltetrahydrofolate
substrate. One possibility is that PurF’ acts an ammonia- present [28], a purN gene is not expected, as PurN
dependent phosphoribosylpyrophosphate aminotransfer- requires 10-formyltetrahydrofolate as the one-carbon
ase, a hypothesis that will require experimental testing. donor. Although superficially similar to 10-formyltetra-
We identified an additional C-terminal domain in hydrofolate, N10-formyltetrahydromethanopterin is not
Methanospirillum hungatei’s PurF. This C-terminal believed to be functional as a formyl group donor [29].
domain is a HEPN-type domain, which might be GAR formyltransferase activity appears to be provided
involved in nucleotide binding. This feature was unique by PurN in the Thaumarchaeota, Thermoplasmata,
to M. hungatei, and its specific function in this PurF Methanomicrobiales, Methanosarcinales, Thermopro-
candidate is unclear. teales, and Halobacteriales. The residues involved in cat-
alysis in the E. coli enzyme (His108 and Asp144 [30])
Glycinamide ribonucleotidesynthetase (EC 6.3.4.13,GARS, are conserved in each of these archaeal enzymes. Metha-
PurD) nosarcina barkeri has been previously determined to
Glycinamide ribonucleotide synthetase, PurD, is an contain tetrahydrofolates [31], which would be required
ATP-grasp protein, responsible for conversion of 5- for PurN activity. Tetrahydrofolates are expected to be
phospho-b-D-ribosylamine (PRA) to 5-phosphoribosyl- present in all PurN-containing organisms. The incor-
glycinamide (GAR), with incorporation of glycine and poration of labeled acetate at C8 of purine synthesized
hydrolysis of ATP to ADP. Transfer of the unstable in M. hungatei and M. barkeri is consistent with this
PurD substrate, PRA, from PurF is believed to occur formyl group being transferred from 10-formyltetrahy-
through a transient interaction between these two drofolate by a PurN-type GAR formyltransferase [32],
enzymes [25]. Studies of PurF and PurD from Aquifex consistent with the presence of purN in the genomes of
aolicus indicated that coupling occurs, but the measured these organisms.
in vitro efficiency was too low to be biologically useful, The nine species of Halobacteria studied have a PurN-
suggesting that another protein or small molecule may like enzyme fused to a PurH1-like domain (which would
be required to reconstitute a functional assembly [26]. also require tetrahydrofolates). Some of these organisms
A gene encoding a protein similar to PurD was identi- have previously been shown experimentally to contain
fied in all purine-synthesizing archaea. Two candidates tetrahydrofolates [33]. H. walsbyi has both the purN-
for PurD were present in Haloarcula marismortui purH1 fusion and a purT-like gene. It is unknown
(rrnAC1109 and rrnAC0307) and Halorubrum lacuspro- whether both genes encode functional enzymes, provid-
fundi (Hlac_1295 and Hlac_1553). We suspect ing H. walsbyi with two alternatives depending on
rrnAC1109 and Hlac_1295 encode the functional PurD growth conditions (as occurs in E. coli), or whether one
enzymes, based on problems with Hlac_1553 and gene is non-functional. The purT gene seems likely to
rrnAC0307, including missing Pfam GARS_N sequences be a recent acquisition by horizontal gene transfer, as H.
and Mg2+ binding sites. These two “extra” PurD-like walsbyi’s closest relatives have only PurN for GAR
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formyltransferase activity, and the H. walsbyi PurT We could not identify a GAR formyltransferase (purN/
sequence is more closely related to bacterial PurTs than purT) gene in seven species of Archaea that appear to
to other archaeal PurT sequences. In our consensus have otherwise intact purine biosynthesis pathways. The
phylogenetic tree for PurT (Additional File 2), the H. two Archaeoglobi, four Methanomicrobiales, and
walsbyi protein was a deep branch near the center of Methanopyrus kandleri have no genes with high similar-
the unrooted tree. ity to known purT or purN genes despite otherwise
The PurT enzyme (previously identified in bacteria complete or near-complete purine biosynthesis path-
[4]) provides GAR formyltransferase activity in archaea ways. Archaeoglobus fulgidus and Methanothermobacter
that have no tetrahydrofolates, including as Methanocal- thermoautotrophicus are known to contain methanop-
dococcus jannaschii, Methanococcus maripaludis, and terin-related folate analogues rather than tetrahydrofo-
Pyrococcus spp. A purT-like gene is also present in the lates, so the unidentified enzymes might use formate as
Sulfolobales, which are believed to have very low levels a carbon source (as in PurT), or may use an alternate
of tetrahydrofolates [33] along with a folate analogue non-folate carbon source other than methanopterin, as
with characteristics of both tetrahydrofolate and metha- discussed above. Although Methanosphaera stadtmanae,
nopterin [34]. The active site residues of the putative Methanobrevibacter smithii, and Methanobrevibacter
archaeal PurT enzymes are identical to those identified ruminantium are intestinal commensals and thus might
in E. coli, with complete conservation at positions 114, rely on an environmental source of purines (despite the
155, 160, 162, 195, 203, 267, 279, 286, 355, 362, and 363 presence of an otherwise complete pathway), M. thermo-
(E. coli numbering) [35]. autotrophicus is an established purine prototroph [39]
In E. coli, the function of PurT for purine biosynthesis and so must have some means to catalyze the GAR for-
requires the presence of formyltetrahydrofolate hydro- myltransferase reaction. Labeling studies are inconsistent
lase, PurU, to provide PurT with formate from 10-for- as to the substrate of the missing GAR formyltransferase
myltetrahydrofolate. Thus, E. coli deletion mutants purN in the Methanomicrobiales. In the presence of formate,
purT and purN purU were unable to convert GAR to M. stadtmanae has been shown to derive C8 from C2
FGAR, while single gene deletions did not block produc- of acetate (most consistent with the use of a tetrahydro-
tion of FGAR [36]. In E. coli, both GAR formyltransfer- folate C1 carrier), while closely related M. smithii does
ase variants – the PurN-catalyzed reaction using 10- not derive C8 from acetate [32].
formyltetrahydrofolate and the PurT-catalyzed reaction
using formate from PurU – require 10-formyltetrahy- Phosphoribosylformylglycinamidine synthetase (EC
drofolate. In those archaea with only archaeal folate ana- 6.3.5.3, PurL, PurS, PurQ)
logues (such as methanopterin), purine biosynthesis Phosphoribosylformylglycinamidine synthetase (FGAM
must proceed differently. Although a PurT enzyme may synthetase or FGAR aminotransferase) catalyzes the
be functional in these organisms, its formate cannot be synthesis of FGAM from FGAR, glutamine, and ATP.
supplied by PurU. FGAM synthetase has been observed to occur as either
A PurU homologue is absent from nearly all of the a single multidomain enzyme (lgPurL) or as a multipro-
PurT-utilizing archaea. These archaea apparently have tein complex composed of a shorter PurL (smPurL) cor-
enough formate present not to require channeling from responding to the central domain of lgPurL and two
a PurU enzyme. We did identify a PurU-like protein in other proteins: PurQ, a glutaminase similar to the C-
each of the Halobacteria and Thaumarchaeota, with terminal glutaminase domain in lgPurL, and PurS,
conserved catalytic residues His 106 and Asp 142 which is structurally similar to the N-terminal domain
(human numbering) [37]. The presence of PurU in these of lgPurL [40-42]. The PurS protein is required for pur-
species, given that only Haloquadratum walsbyi has a ine biosynthesis in B. subtilis [5] and presumably in
candidate for PurT, was surprising. This PurU-like pro- other organisms with smPurL. PurS has been proposed
tein may regulate levels of folates, as proposed for E. to be a “protein-protein interaction module” [43], but
coli [38], or may produce formate for another purpose there is disagreement in the literature about its physio-
in these archaea. A purine-related role is suggested by logically relevant form [40]. Most of the purine-bio-
the genomic location of the purU-like gene in some of synthesizing archaea we studied had genes encoding
the Halobacteria, where it was found in a gene cluster smPurL, PurQ, and PurS, similar to the situation in
with purS, purQ, and purC. The two Thaumarchaeota Gram-positive bacteria.
may be using PurU to produce formate for PurP (dis- In M. labreanum, a large gene encoding a protein
cussed below), but the Halobacteria do not contain similar to lgPurL, Mlab_0300, is present. This gene is
PurP, and thus there is no clear reason for a formate- more closely related to the lgpurL genes in Clostridia
producing enzyme’s gene to cluster with purine bio- and other bacterial species than it is to any gene in the
synthesis genes in the Halobacteria. sequenced archaea. Separate genes for purS and purQ
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are not present in M. labreanum, as would be expected hydrolysis of ATP to ADP. PurM is structurally and
for an organism with a lgPurL. This result is consistent mechanistically related to the central domain of PurL
with the gene being horizontally transferred from Clos- (smPurL), the enzyme in the previous step of purine
tridia after divergence of M. labreanum from its nearest biosynthesis [44,45]. We identified one candidate gene
sequenced relatives. We found Mlab_0300 alternately for purM in each of the purine-biosynthesizing organ-
annoted as “carbamoyl-phosphate synthetase, large sub- isms studied, with bidirectional best hits on E. coli
unit” or FGAM synthase in the databases consulted. We PurM. Three previously proposed [46] active site resi-
did not see compelling evidence for a carbamoyl phos- dues, His190, Asp65, and Asp94 (E. coli numbering), are
phate synthetase assignment for this gene. 100% conserved in the species studied, while replace-
Methanospirillumhungatei,Methanoculleusmarisnigri, ment of His 247 with asparagine occurs in Caldivirga
Methanocellasp.RC-I, Methanoregulaboonei,Methano- maquilingensis, Thermoplasma acidophilum, Picrophilus
coccusvannielii,andMethanococcusmaripaludisencodea torridus, andThermoplasma volcanium . As there is no
formofPurL thatislonger than the othersmPurLs seen other obvious candidate for PurM in these species, we
inmostarchaealspecies.Thelargergeneproduct(which suspect that the H247N change does not significantly
wecallmdPurL)hasapproximately200additionalamino impair the ability of this enzyme to catalyze the reaction.
acidsattheN-terminus,whereaPurS-likedomainwould
occurinlgPurL.Inthesespecies,theN-terminalsequence Conversion of AIR to CAIR (AIR carboxylase, 4.1.1.21, class
sharesonlylowsequenceidentitywiththesimilarportion II PurE; NCAIR synthetase, 6.3.4.18, PurK; and NCAIR
oflgPurLorPurS,butwesuspectthatthisdomainserves mutase, 5.4.99.18, class I PurE)
a similar structural function to PurS or the N-terminal In eukaryotes, a class II PurE enzyme effects formation
portionoflgPurL.FiveofthesixorganismswithmdPurL of 5-amino-4-imidazole-carboxylic acid ribonucleotide
lackapurSgene,supportingthissupposition.M.marisni- (CAIR) directly from AIR by addition of CO [47], with
2
gri has bothmdpurL and a purS gene candidate, inwhat no ATP requirement. However, the conversion of AIR
may be anexample of redundancy. Thepatternof distri- to CAIR in bacteria requires two enzymes, PurK and
butionofmdpurLversussmpurLpluspurSispatchy,with class I PurE. The PurK enzyme uses AIR, bicarbonate,
closelyrelatedspecieshavingdifferentpurLlengths. and ATP to produce the unstable 5-carboxyamino-1-(5-
Thecatalytictriad[41]oftheglutaminase(PurQ,orthe phospho-D-ribosyl)imidazole (N5-CAIR) intermediate
corresponding domain of lgPurL) is present, with the and ADP. The N5-CAIR is then converted to CAIR by a
cysteineandglutamatefullyconserved,andconservationof class I PurE enzyme. Although N5-CAIR is unstable,
histidineinallsequencesexceptthetwoThaumarchaeota, PurE and PurK have not been shown to associate in
whereanasparagineispresent.Wealsoidentifiedanaddi- vitro [6].
tional PurQ-like protein in M. marisnigri, Memar_1841. While a purE gene candidate is present in all purine
Thisputativeproteinlacksroughly150residues,including biosynthesizing archaea studied, the distribution of purK
theallthreeresiduesinthecatalytictriad.Memar_1841is is patchy, as shown in Tables 1, 2, with purK present in
clustered with the candidate genes for purS and purC, most Crenarchaeota, in the Halobacteria, in some Ther-
whilethefull-lengthpurQ,Mmar_1628,isadjacenttothe mococci, and in the Thermoplasmatales. The absence of
geneencodingthemdPurLcandidate.Wesuspectadupli- purK from the methanogens, Ignicoccus hospitalis, and
cationeventorhorizontalgenetransfer(HGT)followedby D. kamchatkensis is consistent with high availability of
gene decay, which would serve to explain both the extra CO in these species’ environments. For those organ-
2
fractionalpurQandanunexpectedpurSgene. isms that do contain a PurK enzyme, we found the
PurS proteins are typically less than 100 amino acids active site residues identified by Thoden and coworkers
in length. We had problems with earlier versions of sev- [48] to be well conserved. Lys 120 (E. coli numbering),
eral databases with finding PurS-like ORFs, which which contacts the adenine ring of ATP, is 100% con-
necessitated searching for missed ORFs with TBLASTN served in archaeal PurK. Glu 153, another adenine-con-
(data not shown). The problem of unidentified ORFs tacting position, is fully conserved except for two cases
appears to be resolved for PurS, but serves to illustrate where it is substituted with glutamine and one case with
the danger of using arbitrary minimum lengths as a cut- aspartate, both fairly conservative substitutions. Arg 80,
off in coding sequence identification. We may be miss- which contacts the ATP phosphates, is present in the
ing additional small but important proteins. Crenarchaeota, but conservatively substituted with lysine
in Thaumarchaeota and PurK-containing Euryarchaeota.
Phosphoribosylformylglycinamidine cyclo-ligase (AIR Gln 11, proposed to contact the AIR substrate, is fully
synthetase, 6.3.3.1, PurM) conserved after correcting for an incorrect translation
PurM, aminoimidazole ribonucleotide (AIR) synthetase, initiation site in Halobacterium sp. NRC-1. An insertion
catalyzes the conversion of FGAM to AIR, with in the Crenarchaeota and generally low homology in the
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Table 1Annotation errorsidentified, by gene
purgenes
F D N T La Sa Qa M K E C B H1c P P2d H2 O
Proteinnameerrors
Partialmisannotation/over-attribution 3 2 1 b
Inappropriatelyvaguename 23 1 1 27 1 4
Notjustifiedduetomissingfeatures 14 2 1 4
E.C.numbererrors
Oneormoremissing 6 13 23 35 1
Oneormoreincorrect/unjustified 14 2 2 2 2 1 1 31 2
Genestructureerrors
Startcodonmis-called 3 1
Pseudogenelabelunjustified 1
Genesymbolerrors
Incorrectgenesymbol 2 1 1 1
Numberofgenesexamined 72 60 31 23 58 51 58 58 28 59 63e 60 13c 46e 31 2f 25
aThethreegeneproductsshareanECnumber.
bNamingofPurEisproblematic.SomePurEsarenotclearlyclassIorclassII,andsomeorganismslackaPurK,makingaclassIItypenamemoreappropriate
evenwhenPurEappearsclassI.WecountedeitheraclassIorclassII-typenameascorrectinthisanalysis.
cHalobacteriafusionsofPurNandPurH1arecountedunderPurN.
dSeparatecountsweremaintainedforPurP-likeproteinsinclusterII.WepreferredgenericnamesandnoECnumber,givenalackofdemonstratedfunctionfor
proteinsinthiscluster.
eAsplitorframe-shiftedgenewascountedasonegene.
fExcludesfull-lengthPurH,countedunderPurH1.
region of Glu 49, thought to make contacts to the ribose al. [51], as previously described for the A. fulgidus pro-
ring of AIR, prevented us from making a definitive tein. The purE genes appear to have been horizontally
determination about conservation of this residue. Lys transferred from a eukaryote, with loss of the ancestral
307, Arg 313, and Lys 314, postulated to contact AIR, organism’s class I purE, as the present genes encode
and Asp 127, postulated to act as a base during the proteins more closely related to eukaryotic PurEs than
reaction, were fully conserved. to any other archaeal PurEs.
Theremay beanevolutionaryadvantagefortheuseof The four Methanobacteria (M. smithii, M. thermoau-
class II PurE or non-enzymatic production of N5-CAIR, totrophicus, M. ruminantium, andM. stadtmanae) have
with avoidance of ATP hydrolysis by PurK, but only in a gene encoding two fused PurE-like domains, but no
thecontextofasufficientlyhighconcentrationofCO to purK gene. The M. thermoautotrophicus and M. smithii
2
giveareasonablerateofCAIRsynthesis.E.coliPurEhas purE-purE’ genes complement both purE and purK dele-
beenshowntocatalyzeconversionofAIRtoCAIR(with- tions in E. coli [52], suggesting that a purK gene is not
out PurK) in the presence of high concentrations of necessary in M. thermoautotrophicus or M. smithii. The
bicarbonate[6].IthasbeensuggestedthatPurKmaynot first domain (PurE) has been characterized as class I
be requiredforhyperthermophilic organismsgrowingat based on amino acid sequence [51]. Intriguingly, a dele-
highCO concentrations,wherethenon-enzymaticcon- tion in the second half of M. smithii gene (the purE’
2
versionofAIRtoN5-CAIRmaybesufficient[49].Patrick region) removed the ability of that gene to complement
et al. have demonstrated complementation of the Keio either purE or purK deletions in E. coli [52], suggesting
collection E. coli purK deletion with over-expression of that it may actually be the PurE’ that is involved in cata-
plasmid-encodedPurE,whichtheysuggesthelpsshiftthe lysis of both steps of this reaction, despite lower similar-
equilibriumpositionofthenon-enzymaticreaction,com- ity of this second domain to known PurE enzymes.
pensatingforlackofPurK[50].Wethusdonotconsider Alternately, the PurE’ domain might be structural in
theabsenceofpurKtobeanindicationthatanunknown nature but strictly required for subunit assembly.
gene is involved in the pathway, so much as a reflection Regardless of which domain is responsible for the cata-
ofavarietyofstrategiesforproductionofCAIRdepend- lysis, we conclude that all four of these double-length
ingoncarbondioxideavailability. PurE-like proteins effect the AIR carboxylase reaction
Archaeoglobus profundus and A. fulgidus have a class alone on the basis of the complementation results with
II-like PurE and no PurK. These PurEs from the the M. smithii and M. thermoautotrophicus genes.
Archaeoglobi have the six conserved distinguishing resi- The other methanogens, the Desulfurococcales, and
dues for class II enzymes as proposed by Matthews et some of the Thermococci have a class I-like PurE and
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no PurK. With the exception of a methionine at position conserved or conservatively replaced with Lys, except in
14 (E. coli numbering), which is not conserved in Thermococcus gammatolerans (TGAM_1268), Thermo-
archaea, the other conserved positions in our PurE can- coccus kodakaraensis (TK0432), and M. labreanum
didates match those identified by Mathews and cowor- (Mlab_0676), where the predicted coding sequence ends
kers [51]. These class I-like PurE enzymes are either before this position. Interestingly, the two Thermococci
using high concentrations of carbon dioxide and AIR as have two purC-like genes, so it is possible that the trun-
substrates, are using N5-CAIR produced from non-enzy- cated purC-like gene does not encode active enzyme. M.
matic reactions, or have an alternate enzyme involved in labreanum’s Mlab_0676, however, is the organism’s only
N5-CAIR production. apparent purC, suggesting that SAICAR synthetase
TheHalobacteriastudiedhaveaPurE-likeproteinthat activity may be possible even when PurC is truncated.
we could not unambiguously assign to either class I or We built a maximum likelihood phylogenetic tree for
classII.Aninsertionafterthefirsthelix(relativetotheE. the predicted PurC-like proteins, including a selection of
colistructure),alysineatposition73,andpooralignment eukaryal and bacterial sequences, based only on well-
in the otherwise conserved region at the end of helix 4 aligned regions (Additional File 2). Bootstrap values
result in no match to the consensus sequence of class I were low, thus interpretation of the results merits cau-
orclassII.Astheseorganismshave aseparatepurK-like tion. Neither the Crenarchaeota nor Euryarchaeota were
gene,thesePurEenzymesonlyneedtoperformtheclass monophyletic in our analysis, nor were the selected
I function, which we expect based on these organisms’ eukaryal and bacterial sequences. Within the limitations
occurrenceincool,aerobicenvironments. of the trees built, the history of purC does not seem to
Except for the Desulfurococcales, the Crenarchaeota match the history of the organisms examined. This
have a class I PurE accompanied by PurK. Experimental could be caused by numerous horizontal transfers or by
support for this assignment is available in Sulfolobus sol- distinctly different selective pressures on these genes,
fataricus, in which the purE and purK genes have each such as if some proteins are not actually SAICAR
been previously shown to complement E. coli with the synthetases or have a second activity in addition to SAI-
corresponding deletion, with no ability of the S. solfatar- CAR synthetase. At least two closely related reactions,
icus purE gene to complement E. coli purK deletions or conversion of IMP to adenylosuccinate (catalyzed by
vice versa [53]. High temperature alone thus does not PurA) and synthesis of b-RFSA-P (4-(b-D-ribofurano-
appear sufficient to remove the utility of purK in the syl)-N-succinylaminobenzene 5’-phosphate) from phos-
absence of high carbon dioxide levels. phorylated b-RFH-P (4-(b-D-ribofuranosyl)
hydroxybenzene 5’-phosphate) [58] require very similar
Phosphoribosylaminoimidazole-succinocarboxamide chemistry and may be the proposed additional or alter-
synthetase (EC 6.3.2.6, SAICAR synthetase, PurC) nate functions for some of the proposed PurC proteins.
The purC gene product, SAICAR synthetase, catalyzes The predicted PurC protein in M. hungatei
the conversion of L-aspartate, ATP, and CAIR into (Mhun_1253) is unusual, in that it includes an N-term-
phosphoribosylaminoimidazole-succinocarboxamide inal radical SAM (S-adenosylmethionine) binding
(SAICAR), ADP, and phosphate. PurC is structurally domain similar to CofG [59] (49% identical to M. jan-
and mechanistically related to adenylsuccinate synthase naschii CofG, 60% identical to a suspected FO synthe-
(PurA), which converts IMP, L-aspartate and GTP to tase subunit I in M. labreanum), in addition to a C-
adenylsuccinate, GDP, and phosphate [54]. E. coli PurC terminal PurC-like domain. While we did not find this
has been kinetically characterized [54], and structures of fusion in other archaeal species, the purC-like genes in
PurCs from several species have been reported [55-57] Methanosphaerula palustris, M. boonei, M. labreanum,
or deposited (MJ1592). M. barkeri (Mbar_A3291), M. marisnigri, T. kodakar-
We identified at least one purC-like gene in all pur- aensis (TK0432), and T. gammatolerans (TGAM_1268),
ine-synthesizing archaeal species. Conservation of the each have a gene with a radical SAM-like motif immedi-
substrate-contacting residues identified by Ginder and ately preceding them on the same strand. We also found
coworkers [57] for the E. coli crystal structure with a nearby gene on the opposite strand with a radical
bound CAIR and ADP is high. Glu 179, which contacts SAM-like motif (sometimes annotated as cofH) in sev-
ADP, and Ser 100, Arg 94, Arg 199, and Asp 175, which eral of the Halobacteria. Although the involvement of S-
contact CAIR, are fully conserved. However, Lys 11 and adenosylmethionine in purine biosynthesis has not been
Lys 13, which make contacts to CAIR, are not present proposed, S-adenosylmethionine is required for thiamine
in the Halobacteria or in the Archaeoglobi. Gln 69 is biosynthesis, which uses the purine biosynthetic inter-
not conserved in the archaea, but a highly conserved mediate AIR. S-adenosylmethione is also required for
histidine is present at the corresponding position in the the conversion of GTP to the FO cofactor. Thus, this
archaeal proteins. CAIR-contacting residue Arg 215 is radical SAM-binding motif might represent a regulatory