Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria

24 Pages·2003·0.99 MB·English

FEMSMicrobiologyReviews27(2003)593^616
www.fems-microbiology.org
Alcohol dehydrogenases from thermophilic and
hyperthermophilic archaea and bacteria
Helia Radianingtyas a, Phillip C. Wright b;(cid:1)
a DepartmentofChemicalandProcessEngineering,SchoolofEngineeringandPhysicalSciences,Heriot-WattUniversity,Riccarton,
EdinburghEH144AS,UK
b BiologicalandEnvironmentalSystemsGroup,DepartmentofChemicalandProcessEngineering,UniversityofShe⁄eld,MappinStreet,
She⁄eldS13JD,UK
Received2December2002; receivedinrevisedform3May2003; accepted12May2003
Firstpublishedonline1July2003
Abstract
Manystudieshavebeenundertakentocharacterisealcoholdehydrogenases(ADHs)fromthermophilesandhyperthermophiles,mainly
tobetterunderstandtheiractivitiesandthermostability.Todate,thereare20thermophilicarchaealand17thermophilicbacterialstrains
known to have ADHs or similar enzymes, including the hypothetical proteins. Some of these thermophiles are found to have multiple
ADHs, sometimes of different types. A rigid delineation of amino acid sequences amongst currently elucidated thermophilic ADHs and
similarproteinsisphylogeneticallyapparent.AllareNAD(P)-dependent,withoneexceptionthatutilisesthecofactorF instead.Within
420
the NAD(P)-dependent group, the thermophilic ADHs are orderly clustered as zinc-dependent ADHs, short-chain ADHs, and iron-
containing/activated ADHs. Distance matrix calculations reveal that thermophilic ADHs within one type are homologous, with those
derived from a single genus often showing high similarities. Elucidation of the enzyme activity and stability, coupled with structure
analysis, provides excellent information to explain the relationship between them, and thermophilic ADHs diversity.
5 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Enzymeactivity; Thermostability; Proteinstructure; Directedevolution; Rationaldesign
Contents
1. Introduction .......................................................... 593
2. ADHs from thermophilic bacteria and archaea ................................ 599
2.1.Zinc-dependent thermophilic ADHs...................................... 600
2.2.Short-chain thermophilic ADHs ........................................ 606
2.3.Fe-containing/activated thermophilic ADHs................................ 607
3. Structures of thermophilic ADHs........................................... 610
4. Conclusions and perspectives .............................................. 612
Acknowledgements ......................................................... 612
References ............................................................... 612
1. Introduction the ability to thrive at high temperatures. By inference,
the internal components must also be able to withstand
Thermophilic and hyperthermophilic organisms have this extreme environment. Thermophiles are microorgan-
isms that grow fastest above 40‡C [1,2]. They are found
within natural and anthropogenic biotopes such as hot
* Correspondingauthor.Tel.: [phone];
waters, sun-heated oils, geothermal areas, heated waste
Fax: [phone].
E-mailaddress: p.c.wright@she⁄eld.ac.uk(P.C.Wright). dumps and cooling waters. Most thermophiles known
0168-6445/03/$22.0052003FederationofEuropeanMicrobiologicalSocieties.PublishedbyElsevierB.V.Allrightsreserved.
doi:10.1016/S0168-6445(03)00068-8
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
5
Table1 9
4
Thermophilesandhyperthermophilesknowntohavealcoholdehydrogenases
Species Origin Topt (‡C) Ae/an Identi¢edproteins Acc.no. Abbv. Ref.
Archaea^Crenarchaeota
AeropyrumpernixstrainK1 hotsedimentandventingwateratacoastal 90 strictae Zn-ADH BAA81251 AePxADHI [69]
(ATCC700893) solfataricvent,Kodakara-JimaIsland,Japan,
1993
longhypothetical BAA80556 AePxADHII [70]
Zn-ADH BAA80235 AePxADHIII
longhypotheticalFe-ADH BAA80973 AePxADHIV [70]
PyrobaculumaerophilumIM2 marinewaterhole,MarontiBeach,Ischia,Italy 100 ae hypotheticalZn-ADH AAL64544 PyAeADHI [71,72]
H
(DSM7253)
.
AAL64366 PyAeADHII R
a
AAL64117 PyAeADHIII dia
n
FE AAL63909 PyAeADHIV ing
M AAL63819 PyAeADHV ty
SR SulfolobussolfataricusP2 volcanichotspring,Italy 75 ae hypotheticalZn-ADH AAK40795 SuSoADHI [17] as,
E7 (ATCC35092) P.C
94 AAK41061 SuSoADHII .W
14-11-03 AAAAAAKKK444111458635779 SSSuuuSSSoooAAADDDHHHIIVIVI right/F
E
AAK42487 SuSoADHVI M
C
y AAK42584 SuSoADHVII S
aan AAK42630 SuSoADHVIII Mic
AAK42635 SuSoADHIX ro
M b
age AAAAKK4422892173 SSuuSSooAADDHHXXIII iolog
n y
ta AAK42983 SuSoADHXIII R
e
NAD-dependentZn-ADH AAK42665 SuSoADHX [64,73] v
G ie
e Sulfolobustokodaiistrain7 Beppuhotspring,Japan 80 ae longhypotheticalZn-ADH BAB65565 SuToADHI [74] w
e s
l (JCM10545) 2
7
Zw BAB65008 SuToADHII (2
art BAB67688 SuToADHIII 003
BAB65032 SuToADHIV )
5
BAB64993 SuToADHV 93
^
BAB67154 SuToADHVI 6
1
BAB67719 SuToADHVII 6
BAB67362 SuToADHVIII
BAB65473 SuToADHIX
BAB64994 SuToADHX
Sulfolobussp.RC3 solfataric¢eld,Pisciarelli,Italy 75 ae NAD-dependentZn-ADH CAA87591 SuRC3ADH [40]
SulfolobussolfataricusMT4 acidichotspring,Agnano,Naples 87 ae NAD-dependentADH NA [39]
(DSM5833)
SulfolobussolfataricusGa solfataric¢eld,Pisciarelli,Italy 75 ae NAD-dependentADH NA [41]
Table1(Continued).
Species Origin Topt (‡C) Ae/an Identi¢edproteins Acc.no. Abbv. Ref.
Archaea^Euryarchaeota
ArchaeoglobusfulgidusVC-16 submarinehotspring,VolcanoIsland,Italy 83 an hypotheticalZn-ADH AAB89145 ArFuADHI [75]
(ATCC49558)
hypotheticalFe-ADH AAB91203 ArFuADHII [75]
AAB90896 ArFuADHIII
AAB89231 ArFuADHIV
Methanothermobacterthermoauto- sewagesludge 60 an hypotheticalpropanediolFe AAB85481 MeThPdDH [76]
trophicus(ATCC29096) dehydrogenase
Methanocaldococcusjanaschii submarinehydrothermalvent; 85 an hypotheticalglycerol-1-phosphate AAB98707 MeJaGDH [77]
H
strainJAL-1(ATCC43067) EastPaci¢cRise dehydrogenase .
R
Methanoculleusthermophilicus applewastedigestor; USA 55 an F420-dependentADH CAA77275 McThADH [78,79] ad
(DSM3915) ia
n
FE PyrococcusabyssistrainOrsay deepseahydrothermalvent 100 an hypotheticalshort-chain CAB49186 PyAbADHI [80] ing
M dehydrogenase/reductase ty
SR hypotheticalFe-ADH CAB50216 PyAbADHII [80] as,
E P
7 hypotheticalthreonine3Zn CAB50292 PyAbThDH [80] .C
94 dehydrogenase .W
14-11-03 P(AyTroCcCocc4u3s58fu7r)iosus volcanicmarinesediment,Italy 100 an NNAADDPP-(dHe)p-denepdeenndtesnhtor1t‡-cFhea-iAnD2‡HADH AAAACC2255555567 PPyyFFuuAADDHHIII [[1881,]81] right/F
E
C O2-sensitive1‡NADP-dependentFe- [45,46,82] M
y andZn-ADH S
aan PyrococcushorikoshiiOT3 marinehydrothermalventatadepthof 95 an longhypotheticalZn-DH BAA29746 PyHoADHI [83] Mic
(ATCC700860) 1395mintheOkinawaTrough,Paci¢cOcean, ro
M b
ag 1994 iolo
en hypotheticalshort-chainDH BAA31026 PyHoADHII [83] gy
ta longhypotheticalFe-ADH BAA29834 PyHoADHIII [83] R
e
G Thermococcushydrothermalis chimneywall,EastPaci¢cRise 85 strictlyan NADP-dependent1‡Fe-ADH CAA74334 THydADH [47] vie
ee AL662 ws
l Thermococcuslitoralis marinethermalspring; Italy,Naples,Bayof 85 strictlyan NADP-speci¢c1‡ADHfragment AAB30263 ThLiADH [46,84] 27
Zw (ATCC51850) Lucrino (2
art Thermococcuszilligii(previously freshwaterhotspring,KuirauPark,Rotorua, 75 an NADP(H)-dependent1‡Fe-ADH AAB63011 ThZiADH [48,85] 003
)
T.AN1)(ATCC700529) NewZealand,1976
5
ThermococcusES-1 blacksmokerchimneypolychaeteworm 91 an sulfur-dependent1‡ADHfragment AAB35052 TES1ADH [46,86] 93
^
(Paralvinellasp.)atJuandeFucaRidge 61
6
Thermoplasmaacidophilum coalrefusepile 60 ae Zn-ADH-relatedprotein CAC12437 TpAcADHI [87]
(DSM1728)
CAC11970 TpAcADHII
ADH-relatedprotein CAC11176 TpAcADHIII [87]
NP394293 TpAcADHIV
Thermoplasmavolcaniumstrain solfatara,VulcanoIsland,Italy 60 an predictedZn-ADH BAB60114 TpVoADHI [88]
GSS1(DSM4299)
BAB59409 TpVoADHV
predictedshort-chainADH BAB59588 TpVoADHII [88]
BAB59216 TpVoADHIII
predictedFe-ADH BAB60449 TpVoADHVI [88]
BAB59540 TpVoADHIV 59
5
5
Table1(Continued). 9
6
Species Origin Topt (‡C) Ae/an Identi¢edproteins Acc.no. Abbv. Ref.
Bacteria^Aqui¢cae^Aqui¢cales^Aqui¢caceae
AquifexaeolicusVF5 thermalsprings, 90 facae hypotheticalZn-ADH AAC07327 AqAeADH [89]
YellowstoneNationalPark,WY,USA
hypothetical1,3-propanediolFe AAC07174 AqAePdDH [89]
dehydrogenase
Bacteria^Firmicutes^Bacillales
Alicyclobacillusacidocaldarius unknown 60 ae NAD-dependentalcohol-aldehyde NA [90]
oxidoreductase
H
GeobacillusstearothermophilusNCAcannedpeas 65 an NAD-dependentZn-ADH BAA14411 BS1503ADH [36] .
R
1503(ATCC12976) a
d
Geobacillusstearothermophilus manurepile 55 facae NAD-dependentZn-ADH CAA80989 BSADH [36] ia
n
FE (DSM2334) ing
M Geobacillusstearothermophilus unknown 70 facan NAD-dependentZn-ADH CAA81612 BSLLD-RADH [37,91] ty
SR LLD-R(NCIMB12403) as,
E P
7 GeobacillusthermoleovoransB23 deepsubterraneanpetroleumreservoirs, 70 ae NAD-dependentshort-chainADH BAA94092 BTlADHI [92] .C
94 Minami-agaandYabaseoil¢elds .W
14-11-03 Bacteria^Firmicutes^Clostridia^Thermoanaerobacteriales^Thermoanaerobacteriaceae NAD-dependentZn-ADH BAB16599 BTlADHII [93,94] right/F
E
Thermoanaerobacterbrockii thermalsprings, 60 an NADP-dependent2‡Zn-ADH CAA46053 ThBrADH [11,13,57, M
C
y HTD4(ATCC53556) YellowstoneNationalPark,WY,USA 60,95,96] S
aan Thermoanaerobacterbrockii hotspring,NewZealand 70 an NADP-linked2‡ADH NA [97,98] Mic
Thermoanaerobacterethanolicus mud,alkalinehotspring; USA,Yellowstone 60 an NADP-dependent1‡Fe-ADH AAG01186 TEJW200ADH [44] ro
M b
ag JW200(ATCC31937) NationalPark iolo
en NADP-dependent1‡Zn-ADH NA [29,30] gy
ta NADP-dependent2‡Zn-ADH NA [29,30] R
e
G Thermoanaerobacterethanolicus algal^bacterialmat,OctopusSpring, 60 an NADP(H)-linkedZn-ADH AAB06720 TE39EADH [25,31, vie
ee 39E(ATCC53033) YellowstoneNationalPark 61,99] ws
l NADP/NADH1‡ADH NA [25,99] 27
Zw Thermoanaerobactertengcongensis hotspring,Tengcong,China 75 an uncharacterisedFe-ADH AAM23605 ThTeADHI [32] (2
art MB4T(JCM11007T) 003
)
AAM23958 ThTeADHIV
5
relatedZndehydrogenase AAM24795 ThTeADHIII [32] 93
^
AAM23957 ThTeADHII 61
6
relatedshort-chainADH AAM23359 ThTeADHV [32]
AAM23376 ThTeADHVI
AAM24694 ThTeADHVII
AAM24982 ThTeADHVIII
AAM25384 ThTeADHIX
Thermoanaerobacterthermohydro- extractionjuicesofbeetsugarfactory 60 an NADP-linked2‡alcohol-aldehyde/ NA [13]
sulfuricus(ATCC35045) ketoneoxidoreductase
Thermoanaerobacterthermohydro- mudhotsprings,NewZealand 60 an NADP-linked2‡ADH NA [97]
sulfuricus(ATCC53016)
H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616 597
ov/ are moderate, and show an upper temperature range of
g
h. growth between 50 and 70‡C [3], although they are still
ef. 00] 00] 01] 7] 02] 02]0]02]02] m.ni able to grow slowly at 25^40‡C. In general, thermophiles
R [1 [1 [1 [9 [1 [1[5[1[1 bi.nl are de¢ned as microorganisms that grow optimally at 50^
c
n 80‡C. Owing to their close phylogenetic relationship to
Abbv. TsElADHI TsElDHII ThMaADHI ThMaADHIIThMaADHIIIThMaADHIVThMaADHVThMaBuDHThMascDH BI(http://www. mmmwieoedsnpeothspri.ahlenTislgehsemeraoamnfydompbtihhceireloiiscrbecimraoelonpdgdreeeasnsreteinlryttaha.etaridvmeaospptaehrdielyt,konmohowodnterawetnietvhitrihnoenra--
C
N Microorganisms that grow better at temperatures of
at 80‡C or above are described as hyperthermophiles [4].
Acc.no. BAC07780 BAC08522 NA NA AAD35386 AAD35497AAD35521AAD35205AAD36001AAD35902AAD35412 onnumber Taatnhudersesesa,olibrnguiattyna.ilssTmohseeyaxrtrheeaamvbeelseboteofengprHios,owlraentdoeodtxomnpaloyitnealntythifairlgo,hmptreewmssaputeerrre--,
essi containing terrestrial and marine high-temperature areas
Identi¢edproteins probableFe-ADH probabledehydrogenase NAD-dependent1‡ADH NADP-linked2‡ADH probableZn-ADH probableFe-ADHFe-ADHprobableFe-butanoldehydrogenaseprobableshort-chaindehydrogenase e;NA=notavailable;Accno=acc [aatbrotm3heengel]igioeeSn.dsrarpsmtmeruhHbsfd[tdieoa5aalyriec]tpnbep.rosthsdeieelFdierrialotpitvioryahcftorer.,oeoltaruhwTkpimanntearhhsidharoroeemycetnprhthroue,ohieeyctnptinirtaplhcdmhhetleeleseiyerolrrytdcesephaostitarhvearhssehereiyneoplmraserdnsrtmvspioieesnetrs,mpuoyegehinssfnsmotreilt,taleofhnipbascmeabtosnlntoermdotw,tttpiahthriitrahcnethiomhnrseteotdrideinwipoecnminoerriaasgoppsitctenhabultfitsnieircsrvhaietoafesloaoatmmotrmirroeatfcwsdtnethlpta.hsiwaantrreheobdiiernlae----l,
v organisms’maximumtemperature,wasstartedintheearly
ati
Ae/an ae ae an an c=facult m1th9ue7sr0masqowupiahtthiilcitcuhsem[d6ic]i.srcoSooivnrecgreaynthoisefmnts,hetihrsmejouinssttteai¢rbeelsdeteibnnyzeytnmhzeeysmfaiencstTfrthhoeamrt-
a
f
T(‡C)opt 50 70 70 80 strictly; mtFhuaernrtmyheoresmntazobyrlmee,,esathneidssoeclaaeptneazdbylmefreoosmfhaactvthienesgaelastomhibcigreoehnotregfmoaunpniesdrmattsuoraebrsee.
=
strictnces. rseoslivsetnantst.tTohweasredrecmomarmkoabnleprfoeatetiunredsenfraotmurathnetsrmanodphoirligcaennic-
e
bic;efer zymes have generated great interest from an industrial
or
er= perspective, especially concerning their potential biotech-
anaRef. nological applications, with many reviews published on
ore; thermophiles and biotechnology (e.g. [7,8]). However, de-
m
ContinuedTable1(). SpeciesOrigin Bacteria^Cyanobacteria^Chroococcales^ThermosynechococcusThermosynechococcuselongatushotspringBP-1 Bacteria^Thermomicrobia^Thermomicrobiales^ThermomicrobiaceaeThermomicrobiumroseumhotspring Bacteria^ThermotogalesFervidobacteriumnodosumthermalspring,NewZealand(ATCC35602)Thermotogamaritimastrainanaerobicmarinemud,MSB8(ATCC43589)VulcanoIsland,Italy,1982 T(‡C)=optimumgrowthtemperaturein‡C;Ae/an=aerobicoptentrez/query.fcgi);Abbv.=abbreviationofdesignatedproteinna szisoamtth[emitett9nhuhhpuy¡lnuoiu,eoteeneeimbO1edtletncsripncse0rr.imenttthdeedeat]isrs,eotoslvi¡aopIealhlthnoinhtaioptewonceiealcdiycnthifugnhhrevtrdsmegitonieleettscricibteehaisroptocohhcbxsreoitreneganremhctetceoaealirtthheeemirnhrnaior¢nnnibyszoaarobamcaieomyigtsrsziscuobfmtedhaoettretoitlfianieoelieceavennaieolsb(dhicwnsciacsaeA,nthlattumtneyrmeoniinDtoytlmosrhsraootitofaenssHeoreaslntsrammBostthtner)hdmhaahigsaeeypnebintaycsipcsuegaticdv,itdtmhlsrhldcrsaiealaeeetotteauybboyimutmmnnobnsoluisA,dezalfidnotoeirsioyicmrDtcet,nefrmlyraeehtsooseoanHa.hlrahttmegnanwrzrheInyshyonrayyteag.ideatttmgssrrvhdaihTrivumtekoosaoerhceeee.bhaenurtrpsgenseimgbipreewvpotobulmonrtnscioeoutiehtalofdtpeaysoseftahsnwhststrnephttuiioisuoboaehlbynsibmudndnictrpidconrlsltoize,iaeirotheetcyr6nahotcrysalyahrm5aliltgdi¢ceibre‡nooneeontoeeocrClndnnodgesr----fl
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
598 H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616
Fig. 1. Phylogenetic tree derived from thermophilic ADH and related protein amino acid sequences extracted from protein databases (Swiss-Prot,
TrEMBL, OWL, PIR, NCBI, PDB, PRF). The sequences were aligned using ClustalW and adjusted by gap insertion before being clustered using the
averagedistancemethodwith1000bootstrap.AbbreviationsareexplainedinTable1.
from di¡erent sources. ADHs play considerable process catalyse lignin degradation [12]. ADHs from thermophiles
andproductionroles,forexampleingenerationofpotable are of interest for industrial alcohol and enzyme produc-
alcohol [11], solvents and acetic acid [12]. ADHs also sup- tion because these microbes are uniquely suited for direct
port the growth of methylotrophs, oxidise alcohols and biomass fermentation to ethanol via reduced-pressure dis-
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616 599
Table2
ThermophilesknowntohaveADHsisolatedfrommarineandterrestrialbiotopes
Species Origin Zn-ADH Fe-ADH scADH F420 ADH
Marinebiotopes
ArchaeoglobusfulgidusVC-16 submarinehotspring 6 6
MethanocaldococcusjanaschiiJAL-1 submarinevent 6
Pyrococcusabyssist.Orsay deepseavent 6 6 6
Pyrococcusfuriosus volcanicmarine 6 6
PyrococcushorikoshiiOT3 marinevent 6 6 6
PyrobaculumaerophilumIM2 marinewaterhole 6
ThermococcushydrothermalisAL662 chimneywall 6
Thermococcuslitoralis marinespring 6
Thermococcussp.ES-1 blacksmoker 6
ThermotogamaritimaMSB8 marinemud 6 6 6
Terrestrialbiotopes
AeropyrumpernixK1 coastalsolfataricvent 6 6
SulfolobussolfataricusP2 hotspring 6
SulfolobussolfataricusMT4 hotspring 6
SulfolobussolfataricusGa solfataric¢eld 6
Sulfolobustokodaii7 hotspring 6
Sulfolobussp.RC3 solfataric¢eld 6
Methanothermobacterthermoautotrophicus sewagesludge 6
Methanoculleusthermophilicus applewastedigestor 6
ThermococcuszilligiiAN1 freshwaterhotspring 6
Thermoplasmaacidophilum coalrefusepile 6 6
ThermoplasmavolcaniumGSS1 solfatara¢elds 6 6 6
AquifexaeolicusVF5 thermalspring 6 6
GeobacillusstearothermophilusDSM2334 mannurepiles 6
GeobacillusstearothermophilusNCA1503 cannedpeas 6
GeobacillusthermoleovoransB23 oilreservoir 6
ThermoanaerobiumbrockiiHTD-4 hotspring 6
ThermoanaerobacterethanolicusJW200 hotspring 6 6
Thermoanaerobacterethanolicus39E hotspring 6
ThermoanaerobactertengcongensisMB4T hotspring 6 6 6
ThermosynechococcuselongatusBP-1 hotspring 6 6
tillation, and yield active thermostable enzymes of com- with a view to future potential for increased stability and
mercial interest [13]. There is also considerable interest in activity for applied purposes.
theuseofsuchenzymesinthechemicalsynthesisindustry,
particularly the pharmaceutical industry, where the pro-
duction of chiral synthons is an increasingly important 2. ADHs from thermophilic bacteria and archaea
stepinthesynthesisofchirallypurepharmaceuticalagents
[14,15]. Thermophilic ADHs are also suitable for synthe- Todate,thereare20thermophilicarchaealand17ther-
sising cofactors NADþ and NADP, used in various pro- mophilic bacterial strains known to have ADHs or similar
cesses [16]. enzymes, including the hypothetical proteins. As can be
Owing to microbial whole genome sequence analysis seen in Table 1, these thermophiles have been isolated
that allows for information on the putative function of a from natural biotopes such as hydrothermal vents, vol-
gene product to be identi¢ed by sequence comparisons, canic marine sediments, hot springs, deep sea chimney
thereisalotofinformationonthermophileswithputative walls and solfataric ¢elds, as well as from anthropogenic
ADHs, complementing studies on experimentally charac- biotopes such as petroleum reservoirs, canned peas, ma-
terised thermophilic ADHs. These data on putative and nure piles, apple waste digestors, beet sugar factories, and
characterised thermophilic ADHs are valuable for further coalrefusepiles.Someofthesethermophilesareknownto
studiesintotheirdiversity.Furthermore,thesedatacanbe have multiple ADHs, sometimes of di¡erent types. For
used to understand more about their stability properties, example, there is one well-characterised and 12 putative
using both directed evolution and rational design ap- ADHs of one type found in Sulfolobus solfataricus [17],
proaches to eventually elucidate structure to function while Pyrococcus furiosus has been found to have two
mechanisms. di¡erent types of ADHs [18]. This is not unusual, consid-
The current study aims to compare and contrast factors ering many organisms contain multiple ADHs and their
that control and a¡ect stability in thermophilic ADHs, physiological roles can sometimes be di⁄cult to disentan-
and how these factors further in£uence enzyme activities, gle [19]. The presence of multiple ADHs within one or-
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
600 H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616
ganism conceivably re£ects the environment in which the pendentADHs,with67%ofthemhavingonlyonetypeof
organism has been exposed and adapted. Therefore, each ADH. This variation might be caused by di¡erences be-
ADH may have a di¡erent role and/or speci¢city for sur- tweenthetwoenvironments,forexamplesalinity,pH,and
vival. nutrients. In their natural habitats, thermophiles form
Several categories of ADHs can be distinguished based complex food webs, consisting of primary producers and
on their cofactor speci¢city, these being: (i) NAD or consumers of organic materials. Their metabolic potential
NADP, (ii) the pyrrolo-quinoline quinine, haem or cofac- includesvarioustypesofaerobicandanaerobicrespiration
tor F , and (iii) FAD. The NAD(P)-dependent ADHs and di¡erent modes of fermentation. Marine biotopes,
420
can further be sub-divided into zinc-dependent ADHs, suchashydrothermalsystemslocatedatshallowandabys-
short-chain ADHs, and iron-activated ADHs [12]. Most sal depth and at active seamounts, are characterised by
of the known ADHs from thermophiles are NAD(P)-de- relatively high concentrations of salt (about 3% v/v) and
pendent, with the exception of the Methanoculleus thermo- by pH values which range from slightly acidic to slightly
philicus ADH that uses cofactor F in the place of alkaline (pH 5.0^8.5) [22]. Thermophiles known to have
420
NAD(P) (Table 1). This similarity of cofactor utilisation ADHsfrommarinebiotopeswereallisolatedfromsimilar
with their mesophilic counterparts indicates that they kindsofenvironments,wereanaerobic,andhaveoptimum
share a common catalytic mechanism [20,21]. growth temperatures ranging from 80 to 100‡C. On the
In order to analyse diversity amongst thermophilic other hand, natural terrestrial biotopes are water-contain-
ADHs and related proteins, all the thermophilic ADH ing volcanic areas [4] such as hot springs, mud holes and
amino acid sequences available from public databases solfataric ¢elds, which are characterised by low salinity
(Swiss-Prot, TrEMBL, OWL, PIR, NCBI, PDB and (0.1^0.5%) and pH values ranging from about 0.5 to 9.0
PRF)1 were compiled and aligned using ClustalW version [23]. Anthropogenic biotopes, such as sewage sludges,
1.8 EBI, for the purpose of constructing a phylogenetic manure and coal refuse piles, however, are more variable
tree using the average distance method and bootstrap environments. Thephysical proximity ofthe organisms, in
analysis. The resulting phenogram (Fig. 1) revealed that addition to their phylogenetic proximity [24], may help to
within the NAD(P)-dependent group, the thermophilic explainwhymostthermophilesfrommarinebiotopeshave
ADHs are orderly clustered according to enzyme type, Fe-ADHswithlittleobservedvarietyamongst them,while
i.e. zinc-dependent ADHs, short-chain ADHs, and Fe- those from terrestrial biotopes mainly have Zn-ADHs and
containing/activated ADHs, irrespective of archaeal or higher variability of ADH types. It is interesting to note,
bacterialorigin,locationofisolationorgrowthconditions. however, that Pyrobaculum aerophilum is the only aerobic
Using this method, ungrouped thermophilic ADHs can be Archaeonisolatedfrommarinebiotopes,andthusitisthe
classi¢ed via phylogenetic relatedness into their respective only species in the group that does not contain Fe-ADH.
ADH types. For example, Sulfolobus tokodaii ADH se- Ontheotherhand,theanaerobicarchaeaMethanothermo-
quences predicted from its whole genome sequence, previ- bacter thermoautotrophicus, M. thermophilicus, and Ther-
ously ungrouped, can be clustered with known Zn-depen- mococcus zilligii AN1 are the only three species isolated
dent ADHs. In this manner, previously ungrouped ADHs from terrestrial biotopes that do not contain Zn-ADHs.
were able to be assigned, allowing for potential extrapola- This ADH variation within thermophiles from di¡erent
tion of associated physiological and structural properties. environments provides evidence that the genetic and met-
It is worth mentioning at this point that ADHs from abolic diversity present in high-temperature environments
closely related species are often clustered together (Fig. re£ects the stress components that characterise the envi-
1). Interestingly, nucleic acid alignments of the coding re- ronments, such as the range of pH, oxidation/reduction
gion from genes encoding thermophilic ADHs that clus- states, solute concentration, gas composition and mineral-
tered together showed a comparable degree of relatedness ogy.
with the amino acid alignments (data not shown),
although analysis of the complete data set of thermophilic 2.1. Zinc-dependent thermophilic ADHs
adh genes did not result in a matching phenogram as the
protein. This result implies that only adh genes with sim- Using the clustering method mentioned above, it can be
ilar functions from closely related species can be clustered observed in Fig. 1 that more than 50% of known thermo-
together. philic ADHs are grouped into the Zn-dependent ADH
Analysis of thermophilic ADH properties in relation to type, dominated bySulfolobus spp. ADHs. When the ami-
phenotypic origin, i.e. terrestrial and marine, shows that no acid sequences of the thermophilic ADHs within the
mostmarinethermophileshaveatleastoneFe-ADH,with Zn-dependent family were compared with the amino acid
50% having more than one type of ADH (Table 2). In sequence from a representative of the mesophilic Zn-de-
contrast, 78% of all terrestrial thermophiles have Zn-de- pendent ADH Zymomonas mobilis (ZyMoADH) (NCBI
accession no. A35260) using Sequence Comparator (v
2.0) after being aligned and adjusted by gap insertion,
1 UptoAugust2002. similarities ranging from 23.6% to 65.4% were observed.
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616 601
Fig. 2. Amino acid alignment ofADHs from T. brockii (ThBrADH), T. ethanolicus 39E(Te39EADH), and T. tengcongensis (ThTeIIADH) in compari-
sonwithC.beijerinckii(ClBeADH).ThesecondarystructuresofClBeADHandThBrADHarealsoshown,togetherwithcatalyticZnþ bindingdomain
(S)andresiduesthatinteractwithligand(c)andmetal(*).Accessionno.ofeachproteinwithinNCBIisavailableinTable1.
This indicates that Zn-dependent ADHs from thermo- and SuToADHIII are the thermophilic Zn-ADHs from
philic and mesophilic prokaryotes are homologous (Table one genus with similarities above 90% respectively. This
3). The comparison also shows that thermophilic ADHs implies that the amino acid sequences, and to some extent
derived from a single genus often show very high the genes responsible for ADH production, are highly
similarity. PyHoADHI and PyAbThDH, ThBrADH, conserved during evolution due to phylogenetic proximity
TE39EADH andThTeADHII, SuSoADHX, SuRC3ADH and thus seemingly necessary for the organisms’ survival.
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart
602 H.Radianingtyas,P.C.Wright/FEMSMicrobiologyReviews27(2003)593^616
Fig. 3. Amino acid alignment of ADHs from G. stearothermophilus strain DSM 2334 (BSADH), NCA 1503 (BS1503ADH), and LLD-R (BSLLD-
RADH) showing conserved region (grey area) and theoretical binding domains for catalytic Znþ (S) and structural Znþ (P). Accession no. of each
proteinwithinNCBIisavailableinTable1.
Many thermophilic ADHs within this group have been ity of ThBrADH are distributed along the whole protein
studied comprehensively ever since Lamedand Zeikus [11] molecule. In addition to that, comparison of ThBrADH
in 1980 successfully isolated a Zn-dependent ADH from with yeast ADH, regarding their catalytic activity and
an anaerobic thermophilic Clostridia Thermoanaerobacter stability in the presence of a number of miscible and im-
brockii (ThBrADH). The enzyme was found to be most miscible organic solvents, revealed that non-polar solvents
active toward 2‡ alcohols in the presence of NADP, and wereabletoenhancethethermalstabilityofbothproteins
retained its activity at high temperature. In a subsequent [28].
experiment, it was found that the amino acid sequence of Another anaerobic thermophilic Clostridia, Thermo-
ThBrADH shares 86.9% similarity with a Zn-ADH from anaerobacter ethanolicus, was found to have two di¡erent
the mesophile Clostridium beijerinckii (ClBeADH), despite types of Zn-dependent ADHs. One was most active to-
the 26‡C di¡erence in thermal stability and the similarity ward 1‡ alcohols, and the other toward 2‡ alcohols
inactivity[25,26].Thisinteresting¢ndingledtoaseriesof [25,29,30]. T. ethanolicus JW200 1‡ ADH, however, had
research studies to investigate the factors that determine preferences toward NAD as a cofactor, while the T. etha-
the thermal stability of ThBrADH in comparison to nolicus 39E 1‡ ADH preferred NADP, which was also
ClBeADH. Peretz et al. [27] proposed that the presence preferred by both 2‡ ADHs. This ¢nding led to the con-
of eight additional proline residues in ThBrADH versus clusion that in nature, T. ethanolicus 2‡ ADHs play a role
ClBeADH (Fig. 2), and the exchange of hydrophilic and in ethanol formation, while T. ethanolicus 1‡ ADHs con-
large hydrophobic residues in ClBeADH for the small hy- vert alcohols to aldehydes [29,30], with di¡erences in the
drophobic amino acids proline, alanine, and valine in hydrophobicclusterspresentintheoverall1‡ADHand2‡
ThBrADH might contribute to the higher thermostability ADH sequences suggesting signi¢cant di¡erences in the
of the T. brockiienzyme. Bogin et al. [26],however, found overall structure of these enzymes [25]. The negatively
thatonlyprolinesubstitutionforSer-24,Leu-316andAla- charged Asp-223, -195 and -215 residues were found to
347 in ClBeADH increased the enzyme’s thermostability, be consistent with the NAD(H) dependence of the horse
while substituting proline for His-222, Leu-275, and Thr- liver, B. stearothermophilus, and Alcaligenes eutrophus en-
149reducedthermalstabilityparameters.Theauthorsalso zymes respectively, whereas the presence of an uncharged
observed that the thermal stability of each chimeric en- residue Gly-198 at the analogous position within the
zyme was approximately exponentially proportional to T. ethanolicus, T. brockii, and C. beijerinckii 2‡ ADH is
the content of the amino acid sequence, indicating that consistent with the NADP(H) dependence of these en-
the amino acid residues contributing to the thermal stabil- zymes [25]. T. ethanolicus 39E Zn-dependent 2‡ ADH
FEMSRE79414-11-03 Cyaan Magenta Geel Zwart