Review Article Reverse Methanogenesis and Respiration in Methanotrophic Archaea

22 Pages·2017·1.03 MB·English

Hindawi Publishing Corporation
Archaea
Volume 2017, Article ID 1654237, 22 pages
http://dx.doi.org/10.1155/2017/1654237
Review Article
Reverse Methanogenesis and Respiration in
Methanotrophic Archaea
PeerH.A.Timmers,1,2,3 CorneliaU.Welte,3,4JasperJ.Koehorst,5 CarolineM.Plugge,1,2
MikeS.M.Jetten,3,4,6andAlfonsJ.M.Stams1,3,7
1LaboratoryofMicrobiology,WageningenUniversity,Stippeneng4,6708WEWageningen,Netherlands
2Wetsus,EuropeanCentreofExcellenceforSustainableWaterTechnology,Oostergoweg9,8911MALeeuwarden,Netherlands
3SoehngenInstituteofAnaerobicMicrobiology,Heyendaalseweg135,6525AJNijmegen,Netherlands
4DepartmentofMicrobiology,RadboudUniversity,Heyendaalseweg135,6525AJNijmegen,Netherlands
5LaboratoryofSystemsandSyntheticBiology,WageningenUniversity,Stippeneng4,6708WEWageningen,Netherlands
6TUDelftBiotechnology,Julianalaan67,2628BCDelft,Netherlands
7CentreofBiologicalEngineering,UniversityofMinho,CampusdeGualtar,4710-057Braga,Portugal
CorrespondenceshouldbeaddressedtoPeerH.A.Timmers;[email protected]
Received3August2016;Revised11October2016;Accepted31October2016;Published5January2017
AcademicEditor:MichaelW.Friedrich
Copyright©2017PeerH.A.Timmersetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Anaerobicoxidationofmethane(AOM)iscatalyzedbyanaerobicmethane-oxidizingarchaea(ANME)viaareverseandmodified
methanogenesispathway.Methanogenscanalsoreversethemethanogenesispathwaytooxidizemethane,butonlyduringnet
methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane
oxidation(i.e.,enzymaticbackflux).NetAOMisexergonicwhencoupledtoanexternalelectronacceptorsuchassulfate(ANME-
1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis
pathway and essential differences between ANME and methanogens are described by combining published information with
domainbased(meta)genomecomparisonofarchaealmethanotrophsandselectedarchaea.Thesedifferencesincludeabundances
and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or
methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively.
Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different
mechanismfordisposalofelectronsandpossiblyarelessversatileinelectronacceptorsusethanANME-2.Futureresearchwill
shedlightonthemolecularbasisofreversalofthemethanogenicpathwayandelectrontransferindifferentANMEtypes.
1.Introduction and ANME-3. The ANME-1 cluster is related to Metha-
nomicrobiales and Methanosarcinales but forms a separate
1.1.AnaerobicMethane-OxidizingArchaea(ANME). Anaer- cluster [2], ANME-2 are related to cultivated members of
obicmethane-oxidizingarchaea(ANME)performanaerobic the Methanosarcinales [4], and ANME-3 are more related
oxidation of methane (AOM) via reversal of the metha- to Methanococcoides spp. [5] (Figure 1). The ANME clades
nogenic pathway. ANME were first discovered in marine arenotmonophyleticwitheachotherandthephylogenetic
sediments where AOM was coupled to sulfate reduction distancebetweenthesubgroupsislarge,with16SrRNAgene
(SR)(Table1,reaction(1)).Here,ANMEformedmetaboli- sequencesimilarityofonly75–92%[6].SubclustersANME-
callyinterdependentconsortiawithsulfate-reducingbacteria 2aandANME-2bformacoherentcladethatisdistinguished
(SRB) that belong to the Deltaproteobacteria [1–3]. Three fromANME-2c[7]andarethereforeoftengroupedtogether
distinct methanotrophic groups were identified: ANME-1 asANME-2a/b(Figure1).Thewidephylogeneticdistribution
(subclusters a and b), ANME-2 (subclusters a, b, and c), isreflectedintheecologicalnicheadaptationofthedifferent
2 Archaea
Methanomicrobiales
ANME-1
Archaeoglobales Methanosaeta
Methanococcales
10%
Methanolobus
AAA Methanohalophilus
GoM-Arc I
ANME-2d
Methanococcoides
ANME-2a/b
ANME-2c
ANME-3
Methanosarcina
Figure1:Phylogenetictreeoffulllengtharchaeal16SrRNAsequencesshowingallmethanotrophiccladessofardescribed(grey)andother
archaeal clades usedinour domainbased(meta)genome comparison(black).The treewas constructedwiththeARBsoftwarepackage
(versionarb-6.0.1.rev12565)[49]using2800sequencesfromtheSILVASSURefNR99database(release119.1)[50].Treeswerecalculated
bymaximumlikelihoodanalysis(RAxML,PHYML)andtheARBneighbor-joiningmethodwithterminalfilteringandtheJukes-Cantor
correction.Resultingtreeswerecomparedmanuallyandaconsensustreewasconstructed.Sulfolobalesasoutgroupwasremovedaftertree
calculations.Thescalebarrepresentsthepercentageofchangespernucleotideposition.
Table1:Gibbsfreeenergychangesunderstandardconditions(Δ𝐺0)foranaerobicmethaneoxidationcoupledtodifferentelectronacceptors
(possibly)performedbyANME.
Reaction Gibbsfreeenergy(Δ𝐺0,kJmol−1)
(1)CH4+SO42−→HCO3−+HS−+H2O −16.3
(2)CH4+4NO3−→HCO3−+4NO2−+H2O+H+ −517.2
(3)CH4+8Fe(OH)3+16H+→CO2+8Fe2++22H2O −571.2
(4)CH4+4MnO2+8H+→CO2+4Mn2++6H2O −763.2
(5)CH4+4/3Cr2O72−+32/3H+→8/3Cr3++CO2+22/3H2O −841.4
ANME clades. ANME clades involvedin sulfate-dependent be coupled to the reduction of different metal (oxides)
AOM (S-AOM) co-occur in many different marine envi- (Table1,reactions(3)–(5)),butlimitedexperimentalevidence
ronments, except for ANME-3 that was mainly found in exists to date that ANME are responsible for this reaction
mud volcanoes and in some seep sediments [6, 8, 9]. In (discussed in Section 3.3). Besides marine environments,
marinesediments,adistinctzonationoccurswhereANME- ANMEinvolvedinS-AOMcanbefoundinterrestrial[25,26]
2a/b dominate upper layers and ANME-2c and/or ANME- andfreshwaterecosystems[27].
1abundanceincreasesindeeperzones,indicatingecological A member of a fourth subcluster, “Candidatus (Ca.)
niche separation [10–15]. ANME also form a versatile part- Methanoperedensnitroreducens,”wasrecentlydiscoveredto
nership with non-SRB such as beta-proteobacteria [16] and perform nitrate-dependent AOM (N-AOM) [28] (Table 1,
Verrucomicrobia[17].ANME,andespeciallyANME-1,have reaction (2)). This cluster was named “ANME-2d” [29] but
also been observed without a (closely associated) bacterial later renamed to “GOM Arc I” [30] and “AOM-associated
partner [5, 12, 13, 18–22]. It was therefore suggested that archaea (AAA)” [6]. Phylogenetic analysis shows that the
ANMEcouldperformAOMalone,usingelectronacceptors ANME-2d cluster is monophyletic with “Ca. M. nitrore-
such as metal oxides, or perform other processes such as ducens” andotherAAA/GoMArcIsequences,butdistinct
methanogenesis [23, 24]. Indications exist that AOM can fromotherANME-2subclusters(Figure1).ANME-2dwere
Archaea 3
initially enriched in bioreactors inoculated with freshwater et al., 2010 [41]. We included both ANME-1 metagenomes
samples[28,31–33].AsANME-2darchaeawereonlyrecently to analyze the organization of genes for the formaldehyde-
recognized,theirenvironmentalpreferencesremaininsuffi- activatingenzyme(TableS2)andtheiron-onlyhydrogenase
ciently studied. So far they have been found in freshwater (TableS3).
canals [31], soils and rice paddy fields [34–36], lakes and
rivers[35],andwastewatertreatmentplants[33].InsituAOM
2.ReversaloftheMethanogenesisPathway
activity of ANME-2d was determined recently for the first
time[36].MoreANMEphylotypesindifferentenvironments 2.1. The Central Methanogenesis Pathway. ANME are de-
andpossiblynewarchaealcladesinvolvedinAOMmayyet scribed to perform “reverse methanogenesis” [44] which
have to be discovered. For example, methyl coenzyme M impliesthecompletereversalofmethanogenesisfromH2and
reductase A genes (mcrA) from Bathyarchaeota (formerly CO2,thatis,hydrogenotrophicmethanogenesis(forkinetic
known as Miscellaneous Crenarchaeota Group) and from and thermodynamic considerations, the reader is referred
the new archaeal phylum Verstraetearchaeota were recently to [45]). During “forward” hydrogenotrophic methano-
found,indicatingtheirinvolvementinmethanemetabolism genesis, CO2 is reduced to CH4 with reducing equiva-
[37,38]. lents derived from H2 (Figure 2). During methylotrophic
This review focusses on archaea performing AOM methanogenesis, this pathway is already partly reversed.
through the reversal of the methanogenesis pathway. We Methylotrophicmethanogensutilizeone-carboncompounds
describe the reversibility of the central methanogenic path- suchasmethylamines,methanolormethylatedsulfurcom-
way,includingthekeyenzymeinmethanogenesisandanaer- pounds (methanethiol, dimethyl sulfide) that are activated
obic methanotrophy (i.e., methyl coenzyme M reductase, to methyl coenzyme M. About 75% of the methyl coen-
Mcr). The possibility of methanogens to perform methane zymeM(CH3-CoM)isreducedtoproduceCH4 andabout
oxidationandofANMEtoperformmethanogenesisisalso 25% of CH3-CoM is oxidized to CO2 using the methano-
addressed.Lastly,thephysiologicaladaptationsofANMEto genesis pathway in reverse during methylotrophic growth.
performrespirationusingdifferentelectronacceptorsduring The oxidative part provides reducing equivalents that are
AOMarediscussed. neededforthegenerationoftheprotonmotiveforceinthe
methanogenic respiratory chain and the reduction of CH3-
1.2. ANME versus Methanogens: Domain Based (Meta)Ge- CoMbymethylcoenzymeMreductase(Mcr)[46](Figure3).
nome Comparison. In order to find additional differences Inallmethanogens,theMcrreactionoperatesintheforward
between archaeal methanotrophs and related methanogens reaction and yields methane and the heterodisulfide of
that could validate and complement findings in the lit- coenzymeBandcoenzymeM(CoB-S-S-CoM):
erature, we performed domain based (meta)genome com-
parison between selected metagenomes of ANME and CH3-CoM+CoB-SH󳨀→CH4+CoB-S-S-CoM;
genomes of methanogens, as done previously for bacterial (1)
genomes [39]. For archaeal methanotrophs, we used the Δ𝐺0 =−30kJmol−1[52]
metagenomes of ANME-1 [40, 41], ANME-2a [42], and
ANME-2d [28, 43]. For methanogens, we used genomes of The heterodisulfide is a central intermediate and acts
closelyanddistantlyrelatedspeciesabletoperformacetoclas- as terminal electron acceptor in all methanogens. In
tic methanogenesis (A: Methanosaeta concilii GP6), methy- hydrogenotrophic methanogens without cytochromes, it is
lotrophic methanogenesis (M-1: Methanococcoides burtonii theelectronacceptorofthecytoplasmicelectron-bifurcating
DSM6242, M-2: Methanolobus tindarius DSM2278, and M- CoB-S-S-CoM reductase (HdrABC) and F420-nonreducing
3: Methanohalophilus mahii DSM5219), hydrogenotrophic hydrogenase(MvhADG)complex[47,48]thatisneededto
methanogenesis (H-1: Methanospirillum hungatei JF-1, H-2: provide reduced ferredoxin for the first step in methano-
Methanobacterium formicicum DSM3637, H-3: Methanococ- genesis; the reduction of CO2 to a formyl group. Within
cus maripaludis C5, and H-4: Methanoregula formicica the methanogens with cytochromes, only a few members
SMSP),andbothacetoclasticandmethylotrophicmethano- of the genus Methanosarcina are able to grow on H2/CO2.
genesis(AM:MethanosarcinaacetivoransC2A).Thegenome They use a ferredoxin-dependent hydrogenase (Ech) for
ofasulfate-reducingarchaeonthatcontainedmostenzymes ferredoxin reduction and an additional membrane bound
formethanogenesisexceptforMcr(S:Archaeoglobusfulgidus methanophenazine-dependent hydrogenase (Vho) for H2
DSM 4304) was also included in the comparison. For each oxidation coupled to the reduction of the heterodisulfide
dataset the protein domains were obtained through Inter- bythemembraneboundCoB-S-S-CoMreductase(HdrDE).
ProScan 5.17-56.0 using the TIGRFAM, ProDom, SMART, F420 cycling can be accomplished using the F420-dependent
PROSITE, PfamA, PRINTS, SUPERFAMILY, COILS, and hydrogenase (Frh) and F420H2: phenazine oxidoreductase
Gene3Ddomaindatabases.Resultsoftheanalysisaregiven (Fpo) complex [48] (Figure 2). For methanogens it is of
inTable2andTableS1oftheSupplementaryMaterialavail- crucialimportancethatMcroperatesintheforwardreaction
ableonlineathttps://doi.org/10.1155/2017/1654237.Sincethe to yield methane and the heterodisulfide. If all reactions
ANME-1 metagenome assembled by Stokke et al. 2012 [40] of the methanogenic pathway are reversed such as during
containedmanybacterialgenes,wedidnotrefertothisdata AOM,thepathwayrequirestheinputofenergyandproduces
for the domain based (meta)genome comparison but only electron donors (Figures 4–6). Therefore, during AOM,
used the ANME-1 metagenome described by Meyerdierks an external electron acceptor is needed which makes the
4 Archaea
(meta)genomeis]andANME-2d-urtoniiDSM6242ngateiJF-1(H-1),aeoglobusfulgidus H-3H-4S 120 120 240 120240 360 120120120480110210360360 111 112 242 111 252 224
ofotherarchaea.DomainabundanceineveryMperformingANMEincludeANME-2d-h[28hanosaetaconciliiGP6(A),Methanococcoidesbmethanogens(H)includeMethanospirillumhu(H-4).Thesulfate-reducingarchaeon(S)isArch AAMAM-1M-2M-3H-1H-2 1111112 1111112 2222224 11111122222224 3333336 11111121111112111111244444481111111221222233333393333336 1111111 3111111 4422222 2111111 4223332 4222222
methanotrophsandselectedgenomesE-1-m[41],andANME-2a[42].N-AOnosarcinaacetivoransC2A(AM),MetDSM5219(M-3).Hydrogenotrophic3),andMethanoregulaformicicaSMSP NME-2aANME-2dHANME-2d 111 111 222 111222 333 111111111444111222366333 211 111 222 111 532 222
mesofANMMethamahiiC5(H- MA
Table2:Domainbased(meta)genomecomparisonofselectedmetagenoindicatedbynumbers.S-AOMperformingANMEincludeANME-1-s[40],a[43].Theacetoclastic(A)andmethylotrophic(M)methanogensinclude(M-1),MethanolobustindariusDSM2278(M-2),andMethanohalophilusMethanobacteriumformicicumDSM3637(H-2),MethanococcusmaripaludisDSM4304. InterProIDANME-1SANME-1CentralpathwayMcr,alphasubunit,N-terminalIPR00318342Mcr,alphasubunit,N-terminalIPR01581123subdomain1Mcr,alphasubunit,N-terminalIPR01582322subdomain2Mcr,alphasubunit,C-terminalIPR00904732Mcr,alpha/betasubunit,C-terminalIPR00892464Mcr,alpha/betasubunit,domain2,IPR02268196C-terminalMcr,betasubunitIPR00317912Mcr,betasubunit,C-terminalIPR02267932Mcr,betasubunit,N-terminalIPR02268042Mcr,gammasubunitIPR003178148Mcr,proteinCIPR00768721Mcr,proteinC-likeIPR02632752Mcr,proteinDIPR00390100Mcr,ferredoxin-likefoldIPR0090241265,10-methylenetetrahydromethanopterinIPR01994600reductaseAcetoclasticmethanogenesisCOdehydrogenase/acetyl-CoAIPR00446001synthasecomplexalphasubunitCOdehydrogenase/acetyl-CoAIPR004461134synthasecomplexbetasubunitCOdehydrogenase/acetyl-CoAIPR00448651synthasedeltasubunitCOdehydrogenase/acetyl-CoAIPR016041112synthasedeltasubunit,TIMbarrelCOdehydrogenasebsubunit/acetyl-CoAsynthaseIPR00370432epsilonsubunit
Archaea 5
S 0 0 0 0 0 0 0 1 04 0 2 1 0 7 000 08 0 7000
4
H- 0 0 0 0 0 0 0 0 00 0 0 0 0 1 000 03 0 27000
3
H- 1 0 0 0 0 0 0 0 00 0 0 0 0 0 000 00 0 0000
2
H- 0 0 0 0 0 0 0 1 00 0 0 0 0 0 000 00 0 8053
H-1 0 0 0 0 0 0 0 1 00 0 1 0 0 0 000 09 0 28040
3
M- 6 2 15 2 6 1 2 0 06 0 0 2 1 14 1710 00 2 9044
2
M- 5 2 21 2 6 1 2 4 08 0 1 2 1 8 4420 04 2 42000
1
M- 4 2 12 5 6 1 2 0 04 0 0 0 1 6 1600 01 0 17030
A 0 0 0 0 0 0 0 0 00 0 0 0 0 0 1900 04 6 23053
AM 7 3 12 4 9 2 4 4 015 0 2 0 1 3 3410 030 0 37000
A
d
2
ME- 0 0 0 0 0 0 0 7 317 3 4 0 0 73 2640 06 8 14000
N
A
H
d
d. 2
nue ME- 0 0 0 0 0 0 0 3 20 3 5 0 0 80 1600 06 1 32055
nti N
o A
C
2: a
le E-2
ab M 0 0 0 0 0 0 0 4 36 0 4 2 1 52 2962 215 2 17044
T N
A
M
1
-
ME 0 0 0 0 0 0 0 0 00 0 0 0 0 15 000 514 1 28243
N
A
S
1
-
ME 0 0 3 11 0 0 0 2 332 2 4 1 0 78 1301 164 4 55180
N
A
InterProID IPR006360 IPR021079 IPR008031 IPR010426 IPR012653 IPR012740 IPR026339 IPR016174 IPR010177IPR009056 IPR020942 IPR012286 IPR024673 IPR027594 IPR011031 IPR006457IPR026476IPR001119 IPR003410IPR011635 IPR008454 IPR002035IPR013608IPR006128IPR006129
MethylotrophicmethanogenesisMethyltransferaseMtaA/CmuAMethanol-cobalaminmethyltransferase,BsubunitMonomethylaminemethyltransferaseMtmBTrimethylaminemethyltransferaseDimethylaminemethyltransferaseMtbBTrimethylaminemethyltransferase,MethanosarcinaMethylaminemethyltransferasecorrinoidproteinreductiveactivaseC-typecytochromesDi-haemcytochrome,transmembrane,nitratereductionDoubledCXXCHmotifCytochromec-likedomainClassIIIcytochromeC(tetrahemecytochrome)Tetrahemecytochromedomain,flavocytochromec3(Shewanella)Octahemec-typecytochromeMethanogenesismultihemec-typecytochromeMultihemecytochromeS-layerdomainsS-layerfamilyduplicationdomainSarcinarrayfamilyproteinS-layerhomologydomainCellexportandcelladhesionHYRdomainCARDBdomainCollagen-bindingsurfaceproteinCna-like,B-typedomainvonWillebrandfactor,typeAVWAN-terminalAdhesionlipoproteinAdhesinB
6 Archaea
S 2 01001010 0 0 1 3 0 0 4 21 210 8 0 0 0 0
4
H- 2 02002020 1 0 1 1 0 0 0 00 060 1 0 0 0 0
3
H- 0 00001011 0 0 0 0 0 0 0 00 100 2 0 0 0 0
2
H- 2 00000000 0 0 0 0 0 0 1 00 000 5 0 0 0 0
1
H- 1 01001010 0 0 0 1 0 0 1 90 330 0 0 0 0 0
3
M- 0 01102010 0 0 11 2 0 5 3 00 210 8 0 0 0 3
2
M- 7 01012010 0 19 21 1 0 0 15 40 1300 8 0 0 0 1
1
M- 2 01012020 0 10 7 2 1 0 4 20 300 7 0 0 0 2
A 0 01001010 0 0 0 0 1 0 0 170 000 3 0 0 0 4
AM 7 01001010 0 0 24 2 0 0 2 50 200 27 0 0 0 4
A
d
2
ME- 6 02013020 0 1 8 1 0 0 12 60 61917 2 2 0 1 39
N
A
H
d
d. 2
nue ME- 2 02025130 0 4 9 2 0 0 4 40 2100 2 0 0 0 6
onti AN
C
2: a
able ME-2 3 01012020 1 3 18 6 1 0 52 445 3937 5 0 1 0 17
T N
A
M
1
ME- 0 20000020 2 0 1 4 0 0 77 1120 4250 22 0 0 0 1
N
A
S
1
-
ME 17 22005140 2 0 1 9 3 0 80 1491 50141 27 1 0 0 12
N
A
erProID R008964 R007253R014522R026430R022504R026392R013426R019127R026485 R002508 R017474 R026453 R026371 R019931 R026428 R002102 R016134R002105 R008965R013784R003305 R006633 R010502 R024361 R031768 R008979
Int IP IPIPIPIPIPIPIPIP IP IP IP IP IP IP IP IPIP IPIPIP IP IP IP IP IP
Invasin/intimincell-adhesionfragmentsPutativecellwallbindingrepeat2ArchaeosortaseAArchaeosortaseBArchaeosortaseCExosortase/archaeosortasedomainExosortase,EpsHExosortaseEpsH-relatedArchaeosortasefamilyproteinArtECellwallhydrolase/autolysin,catalyticPEF-CTERMproteinsortingdomainPGF-pre-PGFdomainPGF-CTERMarchaealprotein-sortingsignalLPXTGcellwallanchordomainVPXXXP-CTERMproteinsortingdomainCellulosome-related/DockerinCellulosomeanchoringprotein,cohesindomainDockerindomainDockerintypeIrepeatCarbohydrate-bindingdomainsCarbohydrate-bindingdomainCarbohydrate-binding-likefoldCarbohydrate-binding,CenC-likeCarbohydrate-binding/sugarhydrolysisdomainCarbohydrate-bindingdomain,family9Bacteroidetes-associatedcarbohydrate-bindingoftenN-terminalCarbohydratebindingmodule,xylan-bindingdomainGalactose-bindingdomain-like
Archaea 7
S 0 1 0 0 30 1 0 2 3 0
4
H- 0 0 0 0 00 0 0 0 2 2
3
H- 0 0 0 0 00 0 0 0 2 0
2
H- 1 0 0 0 00 0 0 0 2 0
1
H- 0 0 0 0 00 0 0 0 2 0
3
M- 0 1 0 0 00 0 0 0 2 0
2
M- 0 0 0 1 00 0 0 0 1 0
1
M- 0 0 1 0 00 0 0 0 2 0
A 0 0 0 0 00 0 0 0 1 0
M
A 0 0 1 0 00 0 0 0 2 2
A
d
2
-
E 3 1 2 2 31 1 1 2 3 0
M
N
A
H
d
nued. ME-2 3 2 2 2 41 1 1 2 3 0
onti AN
C
2: a
e -2
l E
ab M 0 1 0 0 00 0 0 0 1 4
T N
A
M
1
-
ME 0 0 0 0 00 1 0 6 3 1
N
A
S
1
-
E
M 0 0 3 3 30 1 0 8 3 1
N
A
erProID R028973 R014006 R004033 R023576 R005614R019963 R022431 R022432 R003773 R020050 R003719
Int IP IP IP IP IPIP IP IP IP IP IP
Menaquinones3-Demethylubiquinone-93-methyltransferaseSuccinatedehydrogenase/fumaratereductase,flavoproteinsubunitUbiE/COQ5methyltransferaseUbiE/COQ5methyltransferase,conservedsiteNrfDfamilyFutalosinehydrolaseCyclicdehypoxanthinefutalosinesynthaseAminodeoxyfutalosinesynthaseMenaquinonesynthesis(chorismatedehydratase&naphthoatesynthase)FOsynthase,subunit2PhenazinesPhenazinebiosynthesisPhzFprotein
8 Archaea
CH4
CoB-S-S-CoM
2H+ HdrDE
Mcr
CoB-SH
MPhH2 CoM-SH
H2 MPh CH3-CoM
Vho Mtr 2Na+
CH3-H4MPT
F420H2
Mer
F420
H2
Frh
CH2=H4MPT
F420H2
Mtd
F420
H2
CH≡H4MPT
H2O
Mch
CHO-H4MPT
Ftr
H2 CHO-MFR
Fdred
+
H Ech Fmd
Fdox
CO2
Figure 2: Hydrogenotrophic methanogenesis in cytochrome containing Methanosarcina barkeri. Black lines represent presence of
conversions.SeeTable3fornomenclature.
reactionthermodynamicallyfavourable(Table1).Thereverse Merviatheformationofmethanolormethylamine[41],as
reaction of Mcr is therefore an essential step in AOM and wasdetectedindeletionmutantsofMethanosarcina[52,53].
is discussed in Section 2.2. The respiratory chain that is Here,CH3-CoMwaspresumablyconvertedtomethanolby
neededforusingdifferentterminalelectronacceptorswillbe a methyltransferaseand subsequentlyto formaldehydeby a
discussedinSection3. methanoldehydrogenase(Mdh).Then,formaldehydewould
5 10
Evidence that ANME use the reverse methanogenesis be converted to N ,N methylene-H4MPT using a fusion
pathway during AOM is derived from metagenomic and protein of the formaldehyde-activating enzyme (Fae) and a
metatranscriptomic analyses. This showed that all genes hexulose-6-phosphate synthase (Hps) [53] (Figure 6). Both
for the (reverse) methanogenic pathway were present and Fae and Hps were found in the ANME-1 metagenome [41]
expressed in ANME-2a [42] (Figure 4) and ANME-2d and metaproteome [40, 51]. However, no genes coding for
[28] (Figure 5). ANME-1 were consistently lacking the enzymes involved in methanol metabolism were detected
geneencodingN5,N10-methylene-tetrahydromethanopterin in these ANME-1 datasets [40, 41, 51] (Table 2), indicating
(H4MPT) reductase (Mer), which is an enzyme needed to that this alternative pathway probably does not occur. The
oxidize methyl-H4MPT during methane oxidation [40, 41, presence of the Fae/Hps fusion protein in ANME-1 during
44,51](Figure6,Table2).Possibleexplanationscouldbethat AOM could also be explained by its involvement in ribose
(1)themergeneispresentbutnotyetdetected,(2)ANME- phosphate synthesis and not in AOM [54]. Indeed, the Fae
1 used a bypass of this step, and (3) Mer was replaced by genedomainsofANME-1werelocatedinbetweenribulose-
astructuralanalogue.Thefirstpossibilityishighlyunlikely. phosphatebindingbarrelandribosomalproteinS5domains
AlthoughnoclosedgenomeofANME-1ispubliclyavailable (Table S2). The third possibility of a structural analogue is
5 10
todate,allANME-1metagenomesconsistentlyonlylackMer mostlikelysinceananalogueofN ,N -methylenetetrahy-
and no other methanogenic genes. The second possibility drofolate(H4HF)reductase(MetF)wasexpressedbyANME-
was proposed previously where ANME-1 uses a bypass of 1duringAOMwhichcouldreplaceMer[40](Figure6).
Archaea 9
Table3:Nomenclature.
Centralmethanogenicpathway
Fmd Formylmethanofuran(CHO-MFR)dehydrogenase
Ftr Formylmethanofuran-tetrahydromethanopterin(H4MPT)formyltransferase
5 10
Mch N ,N -methenyl-H4MPTcyclohydrolase
Mtd F420H2-dependentmethylene-H4MPTdehydrogenase
5 10
Mer N ,N -methylene-H4MPTreductase
5
Mtr N -methyl-H4MPT:coenzymeM(CoM)methyltransferase
Mcr MethylcoenzymeM(CH3-CoM)reductase
Mdh Methanoldehydrogenase
Fae/Hps Fusionproteinofformaldehydeactivatingenzyme/hexulose-6-phosphatesynthase
5 10
MetF N ,N -methylenetetrahydrofolate(H4HF)reductaseanalogue
Electrontransport
Mvh F420-nonreducinghydrogenase
Vho Methanophenazine-dependenthydrogenase
Fpo F420H2:phenazineoxidoreductase
Fqo F420H2:quinoneoxidoreductase
Hdr CoenzymeB-coenzymeMheterodisulfide(CoB-S-S-CoM)reductase
Frh F420-dependenthydrogenase
Ech Ferredoxin-dependenthydrogenase
MePh/MePhH2 Methanophenazine
MQ/MQH2 Menaquinone
Cytb Cytochromeb
Cytc Cytochromec
MHC Multihemec-typecytochrome
Rieske Rieskecytochromebcomplex
Orf7 Pseudoperiplasmicb-typecytochrome
Nar Nitratereductase
Nap Periplasmicnitratereductase
Nrf Nitritereductase
2.2. Methyl Coenzyme M Reductase (Mcr). The enzymatic ∼14nmolminandmgcelldrymass−1.Abouthalfofthecell
reaction of a purified Mcr from ANME has not been mea- dry mass is protein, so the activity for the ANME archaea
sured to date. The key question is whether a methanogenic wouldapproximate1.4to28nmolminandmgprotein−1.To
Mcr can explain the observed in situ AOM rates or if the estimate the activity per mg of Mcr, the proportion of Mcr
Mcr of ANME is structurally altered. There are three main to cellularproteinisneeded. It wasreportedthat7%ofthe
factors to be considered: the kinetic limitations as defined proteinofANMEmicrobialmatsfromtheBlackSea[67,68]
byenzymeproperties(i.e.,half-maximalactivityataspecific and10.4%ofpeptidesfromHydrateRidgemesocosmsisMcr
𝐾𝑀value),thethermodynamicconstraintsoftheenzymatic [51]. As these were no pure cultures, the actual percentages
reaction,andthemaximalorambientrateoftheenzymatic ofMcrinANMEcellsmaybehigher.Transcriptomedatafor
reaction. For the Mcr from Methanothermobacter marbur- ANME-2d[43]showedthatabout19%ofthetotaltranscripts
gensis,kineticparametershavebeendeterminedtoillustrate were derived from the mcr genes indicating (though not
thereversibilityofreaction(1).Inthemethanogenicreaction, demonstrating)ahighMcrcontentinANME-2d.Estimating
the purified Mcr isoform I [55] catalyzes the production of that 10% of the cellular protein would be Mcr, the specific
methane with a 𝑉max of 30Umg−1 and a 𝐾𝑀 of 5mM for activityoftheenzymewouldbebetween14and280nmolmin
CH3-CoM. The same (methanogenic) enzyme was able to andmgMcrprotein−1,whichisupto25timeshigherthan
−1
oxidize methane to CH3-CoM with a rate of 0.0114Umg the measured reverse reaction rate of the M. marburgensis
anda𝐾𝑀formethaneof∼10mM[56]. enzyme (∼12nmol min and mg Mcr−1 [56]). However, the
ToansweriftheobservedAOMratesareinaccordance reversereactionrateoftheM.marburgensisMcrwasdeter-
with the measured methane oxidation rate for the purified mined under nonsaturating substrate conditions and was
McrenzymefromM.marburgensis,theMcractivityduring thereforenotpossibly representingthetruemaximumrate.
AOMisneeded.EstimatesforAOMratesintermsofactivity Nevertheless,bothreversereactionratesareinthesameorder
(percelldrymass)rangebetween<1and20mmolday−1and of magnitude, other than the forward reaction of 30,000–
−1 −1
g cell dry mass [56–66]. This equals an activity of 0.7 to 100,000nmol min and mgMcr . Thus, it seems that the
10 Archaea
CH4
CoB-S-S-CoM
2H+ HdrDE Mcr
CoB-SH
CoM-SH
MPhH2
CoM-SH
H2 MPh CH3-CoM CH3-OH
Mtr 2Na+
Vho
CH3-H4MPT
F420H2
2H+ Fpo Mer
F420
MPh CH2=H4MPT
MPhH2
F420H2
2H+ Fpo Mtd
F420
MPh
CH≡H4MPT
MPhH2 Mch H2O
CHO-H4MPT
Ftr
H
2 CHO-MFR
Fdred
H+ Ech Fmd
Fdox
CO2
Figure3:MethylotrophicmethanogenesisincytochromecontainingMethanosarcinabarkeri.Blacklinesrepresentpresenceofconversions
andredlinesindicatereversalofthehydrogenotrophicmethanogenicpathway.SeeTable3fornomenclature.
Mcr in ANME may have similar catalytic properties as the from (at least) 1.1mM [74], a few mM [57], to even 37mM
methanogenicenzymeandthatthehighamountofMcrper (equivalent to 3MPa CH4) [58]. Thus, high pressure and
mgtotalcellbiomassinANMEmayinpartcompensatesfor thereforehighconcentrationsofmethaneinthenaturalhabi-
theapparentlyrelativelyslowcatalysis. tat accelerate the oxidation rate of methane by Mcr. Future
Considering the thermodynamic constraints, the Gibbs researchtoaccuratelydetermine𝐾𝑀valuesandratesforMcr
freeenergychangeoftheMcrforwardreactionunderstan- atdifferentmethanepartialpressuresishoweverneeded.This
dard conditions is around −30kJmol−1 [56]. Therefore, the mayseemdifficult,butmicrobialactivitymeasurementsatin
reversereactionisendergonicunderstandardconditionsand situmethanepartialpressurewereshowntobesuccessfulin
willnotproceed.However,highmethaneconcentrations(105 thelaboratory[75].
according to reaction (1) [69, 70]) may lead to a favourable ItwassuggestedthattheMcrreactionistheratelimiting
change in the Gibbs free energy in the direction of AOM. step in reverse methanogenesis [56] which is in line with
High methane partial pressure prevails at many habitats the above described challenges. Supporting these findings,
where AOM has been detected. The solubility of methane there does not seem to be a major change in the amino
at atmospheric pressure is only 1.3mM [71]. Consequently, acidstructurethatdetermineswhetherthebackwardsorthe
increasedAOMrateswerereporteduponpressurizingsam- forwardreactionofMcrisfavoured.Aminoacidalignments
plesofdifferentgeographicalorigin[59,60,72,73].The𝐾𝑀 [67]andthecrystalstructureofANME-1Mcr[76]indicated
of Mcr of M. marburgensis for methane was determined at high overall similarity of the methanogenic and methan-
or above 10mM and reported 𝐾𝑀 values of S-AOM varied otrophic enzyme and unambiguously demonstrated that