Microbial desulfonation

21 Pages·1999·0.3 MB·English

by  Alasdair M. Cook

FEMSMicrobiologyReviews22 (1999)399 419
Review
Microbial desulfonation
Alasdair M. Cook a;*, Heike Laue a, Frank Junker b
a FakultaºtfuºrBiologiederUniversitaºt,D-78457Konstanz,Germany
b EstacioŁnExperimentaldelZaidin,ConsejoSuperiordeInvestigacionesCienti¢cas,E-18008Granada,Spain
Abstract
Organosulfonates are widespread compounds, be they natural products of low or high molecular weight, or xenobiotics.
Manycommonlyfoundcompoundsaresubjecttodesulfonation,evenifitisnotcertainwhetherallthecorrespondingenzymes
arewidelyexpressedinnature.Sulfonatesrequiretransportsystemstocrossthecellmembrane,butfewphysiologicaldataand
no biochemical data on this topic are available, though the sequences of some of the appropriate genes are known.
Desulfonative enzymes in aerobic bacteria are generally regulated by induction, if the sulfonate is serving as a carbon and
energy source, or by a global network for sulfur scavenging (sulfatestarvationinduced (SSI) stimulon) if the sulfonate is
servingasasourceofsulfur.ItisunclearwhetheranSSIregulationisfoundinanaerobes.Theanaerobicbacteriaexamined
canexpressthedegradativeenzymesconstitutively,ifthesulfonateisbeingutilizedasacarbonsource,butenzymeinduction
has also been observed. At least three general mechanisms of desulfonation are recognisable or postulated in the aerobic
catabolism of sulfonates: (1) activate the carbon neighboring the C SO bond and release of sulfite assisted by a thiamine
3
pyrophosphatecofactor;(2)destabilizetheC SO bondbyadditionofanoxygenatomtothesamecarbon,usuallydirectlyby
3
oxygenation,andlossofthegoodleavinggroup,sulfite;(3)anunidentified,formallyreductivereaction.UnderSSIScontrol,
different variants of mechanism (2) can be seen. Catabolism of sulfonates by anaerobes was discovered recently, and the
degradationoftaurineinvolvesmechanism(1).Whenanaerobesassimilatesulfonatesulfur,thereisonecommon,unknown
mechanismtodesulfonatetheinertaromaticcompoundsandanothertodesulfonateinertaliphaticcompounds;taurineseems
tobedesulfonatedbymechanism(1).
Keywords: Fermentation; Oxidation; Reduction; Oxygenation; Hydrolysis; Sulfonate; Novelfermentation
Contents
1. Introduction ......................................................................... 400
2. Organosulfonates occurring naturally, their stability and their biosynthesis ........................... 400
3. Xenobiotic sulfonates in the environment .................................................... 401
4. Formalism in the degradation of organosulfonates ............................................. 402
5. Membrane transport ................................................................... 402
6. Aerobes dissimilating sulfonates as sources of carbon and energy .................................. 403
*Correspondingauthor.Tel.: [phone]; Fax: [phone];Email: [email protected]
400
6.1. Hydrolytic desulfonation of aliphatic compounds........................................... 403
6.2. Monooxygenases and desulfonation of aliphatic compounds................................... 404
6.3. Dioxygenases and desulfonation, including hydrolytic and putative reductive reactions ............... 404
6.3.1. Setting the scene for desulfonation of the aromatic ring, largely with naphthalenesulfonates ...... 404
6.3.2. The benzenesulfonate/ptoluenesulfonate dioxygenase system.............................. 404
6.3.3. The degradation of ptoluenesulfonate via the psulfobenzoate dioxygenase system ............. 405
6.3.4. The putative degradation of ptoluenesulfonate via a reductive reaction ..................... 406
6.3.5. The desulfonation of oaminobenzenesulfonate during meta cleavage........................ 407
6.3.6. The hydrolytic desulfonation of paminobenzenesulfonate after ring cleavage ................. 408
7. Fungal peroxidases and arenesulfonates ..................................................... 408
8. Aerobes assimilating sulfonatesulfur ....................................................... 408
9. Anaerobes dissimilating sulfonates as sources of carbon and energy ................................ 410
9.1. Sulfonates as electron acceptors in anaerobic respiration ..................................... 410
9.2. Sulfonates as electron donors in anaerobic respiration ....................................... 411
9.3. Sulfonates in fermentation ........................................................... 412
9.4. Widespread reactions ............................................................... 413
10. Anaerobes assimilating sulfonatesulfur ..................................................... 413
Ackn.o.w.l.e.dg.m..en.t.s......................................................................... 414
Refe.re.n.c.es............................................................................... 414
1. Introduction naturally [19]. The O sulfonates are usually [19],
but not always [20], cleaved hydrolytically by en-
Sulfonated organic compounds, here those with a zymes in EC 3.1.6.- (sulfuric acid hydrolases).
C SO moiety, in£uence our picture of the world N Sulfonates are also known (e.g. N sulfoglucos-
3
around us, whether the words are used ¢guratively amine, representing heparin) and appear to be sub-
or literally. The natural product taurine (Fig. 1) has ject to facile hydrolysis by enzymes in EC 3.10.1.-
roles in vision, neural function and the digestive (acting on sulfur nitrogen bonds). The C sulfonates
tract, amongst others [1]. Many dyestu¡s (Fig. 2), are thermodynamically much more stable [21] and
which add color to e.g. inks, clothes or foodstu¡s, are not subject to chemical hydrolysis (taken as gen-
are sulfonated. As there is now little voluble com- eral knowledge by e.g. [22]). One can thus anticipate
plaint about sulfonated compounds in the environ- a di¡erent biochemistry in the degradation of these
ment, especially after the development of sensitive C sulfonates, compared with the N and O sulfo-
analytical methods (e.g. [2 4]), it seems likely that nates.ThesulfuratominC sulfonateswouldappear
extensive degradation occurs, despite the poor bio- to be in the 5+ oxidation state ([23,24]; cf. [18]).
degradability of at least some sulfonates (e.g. [3,5 Until recently, the naturally occurring sulfonates
8]). This review explores these degradative phenom- were considered to be relatively few in number,
ena,which,overtheyears,writerswithverydi¡erent though often in important functions (e.g. taurine,
backgrounds have approached (e.g. [9 18]). Recent coenzyme M, the plant sulfolipid, sulfolactate and
advances, however, allow a newly rationalized over- methanesulfonate in Fig. 1). It is now clear that or-
view of the ¢eld. ganic matter in soils (e.g. [25]) and in marine sedi-
ments [26] is sulfonated, and determinations of frac-
tions from river samples by pyrolysis-mass
2. Organosulfonates occurring naturally, their spectrometry indicate this to be humic material
stability and their biosynthesis [27]. As humus is subject to biophysical and bio-
chemical alteration [28], including a relatively rapid
Strictly, organosulfonates are those compounds £uxthroughthesulfonatepool[29,30],onecanfairly
containing the SO moiety, so sulfate esters are presume that the number of sulfonated compounds
3
O sulfonates, which have a very wide occurrence occurring naturally is very high. Indeed, biotransfor-
401
Fig.1.Somenaturallyoccurringorganosulfonates.Taurine,discoveredin1836,indicateshowlongbiologistshaveworkedwithsulfonates
[1].Thesulfonate ofmethanogens,coenzyme M,isamuchmorerecentdiscovery(cf.[161]),whiletheimportanceofmethanesulfonate in
the sulfur cycle has only now been recognized [69]. Cysteate was ¢rst observed in biological systems as a weathering product from cys-
teine in wool [162], but it is now better known as an intermediate in one pathway of taurine formation [1] or as a precursor [163] of the
majorsporecomponent,sulfolactate[164].Isethionate,initiallyfoundasamajoranioninsquidnerve[22],isnowknowntohaveawider
distribution [165]. Sulfoacetate is a degradative intermediate [166] from sulfoquinovose in the plant sulfolipid in the thylakoid membrane
[40].Bacterialanddiatomalsulfolipids(notshown)arealsoknown[167,168].Anunde¢ned,butpresumablyaliphaticcompoundisfound
inhighconcentrationsinplasmacellsfromheartsamplesofAscidiaceratodes[23].Incontrasttothesealiphaticcompounds,onlyonede-
¢ned aromatic sulfonate seems to be known, aeruginosin B [169]. Unde¢ned sulfonates in soils, largely in humic materials, and in marine
sedimentsseemtobewidespread[25 27].Humicmaterialischaracterizedbyitsheterogeneity[28],so,asitpresumablyrepresentsamajor
reservoirofnaturalorganosulfonate,itispresentedinthe¢gure,butitcannotbegivenastructure.
mation of these numerous organosulfonates occurs 3. Xenobiotic sulfonates in the environment
naturally [30], so perhaps one should not be sur-
prisedwhenxenobioticcompoundsaredesulfonated. The detergents used to clean clothes, dishes, or
There would appear to be two general routes to £oors, usually contain sulfonated compounds,
generate C sulfonate bonds. One route with many whether as surfactants [17], hydrotropic agents
variations involves oxygenation of cysteine sulfur (p-toluenesulfonate, Fig. 2) or optical brighteners
to the sul¢nate (EC 1.13.11.20), decarboxylation [8]. Some sulfonates are used in molar excess in dye-
(EC 4.1.1.29) and a subsequent oxidation (e.g. EC stu¡ processing (m-nitrobenzenesulfonate, Fig. 2).
1.8.1.3) to yield taurine and a large number of its Yet other sulfonates (naphthalenesulfonates, Fig. 2)
derivatives [1]. Alternatively there is addition of sul- enter the environment in waste streams: chemical
¢te at a C C double bond as in the synthesis of synthesis of sulfonates typically give yields of 80
coenzyme M [31], a variant of which is probably 90% with many sulfonates as by-products [33], and
involved in the synthesis of the sulfoquinovose moi- many of these compounds can be detected in e.g.
ety of the plant sulfolipid [32]. rivers [3] or seepage from waste dumps [34,35]. In
402
addition, it is clear that some sulfonates in the envi- itant excretion of the carbon moiety [44 48]; the
ronment (e.g. ‘alachlor-ethanesulfonate’, Fig. 2) are regulation is a global system for scavenging sulfur
generated biologically from non-sulfonated xenobio- (Section 8). In anaerobes, this strict formalism is
tic compounds [36]. Further sulfonates or deriva- perhaps better known and the di¡erent metabolic
tives, e.g. saccharine (Fig. 2) enter the environment typesareeasilyrecognized; sulfonatesassulfursour-
via foodstu¡s. ces (Section 10), sulfonates as electron sinks in res-
An interest in desulfonation reactions involving piration, sulfonates as electron (and carbon) sources
xenobiotic compounds has been apparent since for e.g. nitrate respiration, and sulfonates as sub-
about 1950 (cf. [37]), though thorough proof of a strates in fermentions (Section 9). These processes
desulfonation reaction was to wait some 15 years, will be examined in turn.
till arguments about the degradation of branched- One process of almost (cf. Section 7) universal
chain alkylbenzenesulfonate surfactants forced direct relevance for the metabolism of organosulfonates is
analyses to be done (cf. [38,39]). Indeed, the non- the transport of the compound across the cell mem-
biodegradability of the ¢rst generation of benzene- brane. It has been largely ignored.
sulfonate surfactants (e.g. [10]) led at least one au-
thortoconsidertheplantsulfolipidasareplacement
[40]. Another 25 years went past before the ¢rst en- 5. Membrane transport
zyme desulfonating a xenobiotic compound was pu-
ri¢ed [41], but the pace of development and the level Organic sulfonic acids are strong acids whose pK
of understanding have increased markedly in the values, if unin£uenced by other substituents, are
meantime. probablynegative[49].Thismeansthatthesulfonate
groupischargedatphysiologicalpHvalues,withthe
consequence that transport is required to bring the
4. Formalism in the degradation of organosulfonates substrate into contact with the cytoplasmic enzymes
involved in its degradation (cf. Section 7 for the re-
Thereappeartobenoreportsoftheaccumulation verse situation).
of naturally occurring sulfonates under anoxic con- Biedlingmaier and Schmidt detected transport sys-
ditions, but until recently it was believed that arene- tems for e.g. ethanesulfonate or taurine in Chlorella
sulfonates were degraded under oxic conditions fusca [50,51] and in cyanobacteria [52] and reported
only [12,42], and desulfonation reactions in anaer- on their kinetics. Thurnheer et al. [53] o¡ered
obes were ignored. The rapidly growing understand- indirect evidence for transport processes in that
ing of anaerobic desulfonation reactions involving the cell membrane of Alcaligenes sp. strain O-1 is
aliphatic and aromatic compounds, and their abun- selectively permeable to isomeric arenesulfonates.
dance, is now illustrated in Sections 9 and 10. Direct transport assays were done by Locher et al.
It is helpful to consider critically the role of the [54] with Comamonas testosteroni T-2 and p-toluene-
organosulfonate in the metabolism of the organism sulfonate. They presented evidence for an inducible,
under study. In aerobes, the sulfonate can be found secondary proton symport system, which was not
as a carbon and energy source only, concomitant su⁄ciently stable to allow biochemical work to be
withexcretionofthesulfonatemoiety.Astheorgan- done.
isms do not grow in the strict absence of sulfate in Geneticevidenceforatransportsystemfortaurine
the growth medium [37,43], it is presumed that the is available in Escherichia coli MC4100 [55]. The
catabolic activities (uptake of arenesulfonate, desul- genes are located in an operon termed tauABCD
fonation and excretion of the sulfonate moiety) are (accessionnumberD85613; [55]),andTauABCcom-
independent of the supply of sulfate for growth, i.e. priseanABC-typetransporterofunknownsubstrate
of a sulfate transport system. In addition, the regu- speci¢city (see also Section 8). Genes for a putative
lation involved is largely induction of a catabolic transport system for methanesulfonate (cf. [56]) are
pathway (Section 6). Alternatively, the sulfonate is under study, and manuscripts are pending (J.C.
used as a source of sulfur for growth with concom- Murrell, personal communication).
403
Fig. 2.Afewxenobiotic sulfonates.Manyofthesecompoundsareverysimple,and, asinthecaseofthenaphthalenesulfonate, represen-
tative for a large number of substituted compounds. The azo dyestu¡, tartrazine, represents a widely used class of dyestu¡s. ‘Alachlor-
ethanesulfonate’ represents a class of compounds formed from chloroacetanilide herbicides, alachlor in this case [36]. More information
onxenobioticsulfonatescanbefoundelsewhere(e.g.[170]).
6. Aerobes dissimilating sulfonates as sources of bond generates the acetate and regenerates the TPP
carbon and energy [57]. The acetate is oxidized to CO and water, with
2
anaplerotic reactions allowing channelling of inter-
Whereas a wide range of compounds has been mediates for biosynthesis [59].
showntoserveasasolesourceofcarbonandenergy The ¢rst known source of sulfoacetaldehyde was
forthegrowthofaerobicbacteria(e.g.[6]),bethisin taurine, but di¡erent organisms use transaminations
pure culture or in (model) sewage, there is much less to di¡erent amino-group acceptors (e.g. EC 2.6.1.55)
information available on the reactions involved, es- (cf.[60])oroxidation(EC1.4.99.2)[61]forthetrans-
pecially on the desulfonations. One can now be sure formation. Recent work shows that the catabolism
that there are hydrolytic desulfonations, desulfona- ofisethionate([62]; cf.[63])andpossiblysulfoacetate
tions inherent in monooxygenations and in dioxy- converge at sulfoacetaldehyde [64], but it is unclear
genations, and a formally reductive desulfonation is how the sulfoacetate is reduced to the aldehyde. The
postulated. enzymes involved in these reactions are found in a
wide range of genera including Pseudomonas [60],
6.1. Hydrolytic desulfonation of aliphatic Acinetobacter [62], Aureobacterium and Comamonas
compounds [64].
Itseemspossiblethatthecatabolismofcysteateor
The ¢rst desulfonation to be characterized was sulfolactate could be channelled through taurine
that of sulfoacetaldehyde, which is hydrolyzed quan- [65,66], but some preliminary enzyme work with cys-
titatively to acetate and sul¢te by a thiamine pyro- teate leads one to suspect the presence of a novel
phosphate (TPP)-coupled lyase (EC 4.4.1.12) from desulfonation [67].
several bacteria (cf. Fig. 6) [57,58]. It is possible to The only other known hydrolytic desulfonation is
hypothesizeanenol-adductoftheTPPwhoseforma- that involved with 4-sulfocatechol, which will be
tion is aided by loss of the sul¢te anion, a good treated with the other aromatic compounds in Sec-
leaving group; addition of water across the double tion 6.3.
404
6.2. Monooxygenases and desulfonation of aliphatic fate was the direct product of desulfonation. Knack-
compounds muss’groupgavemoreexperimentalsupportforthis
idea with a naphthalenesulfonate model [79]. Work
The monooxygenation of n-alkane-1-sulfonates, a with amino- and hydroxynaphthalene-2-sulfonates
family of surfactants, was detected many years ago [80] led to Sphingomonas sp. strain BN6, but despite
[68], but it is only recently that a monooxygenation muchwork(e.g.[81]),thisdesulfonationhasalsonot
reaction, from Methylosulfonomonas methylovora, been examined directly because of the inactivity of
was characterized. The substrate is the natural prod- the oxygenase in cell-free extracts. Indirect evidence
uct methanesulfonate [69,70] and the monooxygen- on the nature of the oxygenase comes from an un-
ase system involved [56,71,72] belongs to the mono- published DNA sequence (accession number
nuclear iron systems better known in the bacterial U65001) located adjacent to the partial DNA se-
oxygenation of aromatic compounds [73]. These en- quence ofa putative1,2-dihydroxynaphthalenediox-
zymes contain an electron transport chain, that col- ygenase (the next enzyme in the degradative path-
lects electron pairs from NADH on a £avin, which way). A comparison of the deduced protein
delivers single electrons via [2Fe 2S] ferredoxin and/ sequence of the ferredoxin component shows high
or [2Fe 2S] Rieske centers to the mononuclear iron sequence similarities with the ferredoxin components
atthereactivecenter(cf.[74])wherethereducediron of naphthalene dioxygenase and of benzene dioxy-
activates the oxygen. Activated oxygen presumably genase.
attacks the carbon atom to yield the unstable hy- Thoseworkingwiththemononuclearironoxygen-
droxy-methanesulfonate, which spontaneously loses asesystemsareusedtocharacterizethemintoclasses
thegoodleavinggroupsul¢tetoformformaldehyde, I,IIandIII,andtodistinguishthemfromthediiron
a typical intermediate in C metabolism [75]. The systems [82 84].
1
three-component system will oxygenate ethane- and
propanesulfonates, but does not accept any further 6.3.2. The benzenesulfonate/p-toluenesulfonate
substitution on the alkane. dioxygenase system
The destabilization of the otherwise inert C SO p-Toluenesulfonate (TS) has turned out to be a
3
bond by the introduction of a second heteroatom to more rewarding substrate as regards elucidation of
the carbon atom, which causes spontaneous loss of desulfonationreactions,andthreedi¡erentpathways
sul¢te, is a mechanism that will be observed many foritsdegradationhavebeendiscoveredorproposed
times is this review. It is presumed to be the basis of (Fig.3).Theinducibledioxygenasewhichcausesring
the formation of oxaloacetate from sulfosuccinate in desulfonation has beenseparatedinextractsofAlca-
Pseudomonas sp. strain B51 [76], though the nature ligenes sp. strain O-1. The reaction catalyzed is:
of the monooxygenase here is still unknown. Anoth-
NADH(cid:135)O (cid:135)TS(cid:135)H(cid:135) !NAD(cid:135)(cid:135)
er monooxygenation is presumed to be involved in 2
the degradation of naphthalene 2,6-disulfonate [77].
4 methylcatechol(cid:135)HSO
3
6.3. Dioxygenases and desulfonation, including
hydrolytic and putative reductive reactions The enzyme is probably a three-component system
that is also benzenesulfonate dioxygenase (desulfo-
6.3.1. Setting the scene for desulfonation of the nating) [85]. This desulfonative enzyme has an in-
aromatic ring, largely with completely explored, broad substrate range which
naphthalenesulfonates involves deaminating o-aminobenzenesulfonate
The bulk of research on desulfonation has been [85,86]. The general phenomenon of spontaneous re-
done with aromatic compounds. Initial work re- actions following oxygenation of the ring is large,
viewed by Cain [9] established the idea that dioxyge- and turns out to be widespread in nature [85,86].
nases destabilized the inert C sulfonate bond and In contrast to the broad substrate range of the en-
that the sulfonate moiety was released as sul¢te zyme, however, induction of the enzyme in strain O-
[38,78], thus correcting some earlier claims that sul- 1 is very speci¢c [85].
405
Fig. 3. Three pathways for the catabolism of p-toluenesulfonate. The uppermost pathway was established in Alcaligenes sp. strain O-1
[85], the middle pathway was established in C. testosteroni T-2 [41,43,90] and the tentative third pathway is derived from data with a
‘pseudomonad’ [38,100]. The desulfonation catalyzed by PsbAC (middle pathway) is analogous to that in the uppermost pathway (see re-
actioninbrackets),but,tosimplifythe¢gure,itisnotshown.
In bacterial nutrition, the combination of utiliza- genases which yield p-sulfobenzoate (PSB). PSB is
tion of TS and benzenesulfonate is widespread desulfonated, as predicted above, via dioxygenation
[37,87 89], so it is hypothesized that an enzyme sys- by a two-component dioxygenase involving reduc-
tem analogous to the TS-dioxygenase system in Al- tase C and oxygenase A (EC 1.14.12.8; a class IA
caligenessp.strainO-1iswidespread.Balashovetal. system) and spontaneous loss of the leaving group,
[88,89] ¢nd the enzyme(s) to be plasmid-encoded. sul¢te, to yield protocatechuate [41,92]:
NADH(cid:135)O (cid:135)PSB(cid:135)H(cid:135) !NAD(cid:135)(cid:135)
6.3.3. The degradation of p-toluenesulfonate via the 2
p-sulfobenzoate dioxygenase system
protocatechuate(cid:135)HSO
The degradation of TS in Comamonas testosteroni 3
T-2 is currently the best understood desulfonative
pathway, though there are still signi¢cant gaps in Reaction stoichiometry and dioxygenation have
our knowledge, especially in the biochemistry and been established [41,43], and the identical oxygenase
genetics of the transport system (Section 5). The ini- component is found in two independent isolates
tialattack(Fig.3)ismonooxygenationofthemethyl [92].
side chain by a two-component monooxygenase sys- Expression of enzyme activity allowed groups of
tem [90,91] followed by two characterized dehydro- co-regulated genes to be determined [93]. The genes
406
encoding the enzymes for the degradation of TS via a class II transposon (cf. [98]), as predicted [94]. A
PSB in C. testosteroni T-2 and of PSB in C. testos- transposition event is observed in conjugation ex-
teroni PSB-4 are located on two conjugative mega- periments involvingstrain PSB-4(donor) andaplas-
plasmids designated pTSA and pPSB, respectively mid-free, psb recipient. The psb genes transpose to
([94]; cf. [13]). The genes encoding degradation of the chromosome with concomitant loss of plasmid
protocatechuate are chromosomal in both organ- pPSB [94]. Currently, the composition of operon
isms. Both plasmids were estimated at 85 kbp, but R3 is speculative, with only psbA (the oxygenase
an expanded data set indicates that pTSA is about component) ¢rmly attributed and psbC (the reduc-
73 kbp [95]. pTSA belongs to the incompatibility tase component) tentatively ascribed; the functions
group IncP1L. It is readily lost under non-selective of the gene products of psbXYZ, open reading
conditions, giving rise to mutant TER-1, which can- frames, are still unknown.
not grow with TS and was very useful for conjuga- The degradation of PSB in C. testosteroni T-2 was
tion experiments to explore the catabolic genes di⁄culttostudyforseveralyears,becausefunctional
[94]. reductaseCwasnolongerfound,anditsactivitywas
The four genes (tsaMBCD) encoding the enzymes complemented by reductase B of the toluenesulfo-
which convert TS to PSB comprise an operon, the nate methyl-monooxygenase (TsaB in Fig. 3). It
tsa operon, that corresponds to regulatory unit R1 was proposed that a mutation in the putative psb
(Fig. 3) [93,94], putatively with the divergently tran- operon had occurred [92,94]. A culture of strain
scribed, LysR-type regulator gene, tsaR (accession T-2 has since been recovered that was stored prior
number U32622) [91]. The intergenic region between to the putative mutational event, and it apparently
tsaR and tsaMBCD shows the characteristic features also encodes the psbAXYCZ structure found in
of LysR-type regulated systems [96] and a putative strain PSB-4, whereas psbXYCZ seem to be missing
sigma70 housekeeping promoter for tsaMBCD is from C. testosteroni T-2 (DSM 6577) [97], thus con-
present [91]. Many newly isolated bacteria, selected ¢rming the hypothesis on a mutation. PSB degrada-
to utilize TS, contain the tsaoperon (shown by PCR tion is ready and widespread [37,88,99], so one won-
andpartialDNAsequence)andshowthesamephys- ders whether the conjugative plasmids and the
iologicalpropertiesasC.testosteroniT-2,namelythe transposition observed represent the transmission
transient excretion of the intermediates p-sulfoben- of one operon, or whether other degradative path-
zylalcohol and PSB during growth with TS [95]. ways remain to be found. The nature and the loca-
This operon, too, seems to be widespread. tion of the transport system, which di¡ers from that
A preliminary sequence [97] is available for the for TS [54], is another open question.
putative psb operon, which corresponds to regula-
tory group R3 (Fig. 3) [93]. The operon structure 6.3.4. The putative degradation of p-toluenesulfonate
in strain PSB-4, psbAXYCZ, is located between via a reductive reaction
two copies of the 3.2-kbp insertion element IS1071 The thirdpathway for the degradation of TS (Fig.
(AccessionnumberM65135),andpossiblyrepresents 3) is generated from the initial identi¢cation of
Fig.4.Thedegradativepathwayoforthanilate(2AS)inAlcaligenessp.strainO-1.Theoxygenationoforthanilatetogenerate3-sulfocate-
chol (3SC) involves an intermediate analogous to that in the upper line of Fig. 3; for simplicity, it is not shown. The product from oxy-
genation(andsubsequentspontaneoushydrolysis)of3-sulfocatecholisshownastherelaxedform(Z,E)of2-hydroxymuconate(2HM).In
fact all isomers can be found, but the most common (99%) is shown. This stresses the importance of spontaneous reactions in the bio-
chemistryofdegradationofaromaticcompoundsingeneral,whereastheemphasishereisonthedesulfonationreaction[85,86].
407
3-methylcatechol [100] and the subsequent identi¢ca-
tion of sul¢te as the primary fate of the sulfonate
moiety [38]. Thus formulated, the pathway repre-
sents a desulfonation that is formally reductive,
even if nothing about its nature is known. The val-
idity of the hypothesis depends on the quality of the
paper chromatographic methodology used to identi-
fytheorganicintermediate.Thehypothesiswouldbe
stronger, if another isolation of the organism were
achieved.
6.3.5. The desulfonation of o-aminobenzenesulfonate
during meta cleavage
The widespread ring dioxygenation with the ac-
companying spontaneous desulfonation and the
reductive desulfonation both occur prior to ring
cleavage. The degradative pathway for o-aminoben-
zenesulfonate (orthanilate) in Alcaligenes sp. strain
O-1 turns out to involve a multi-component dioxy-
genase system which causes stoichiometric deamina-
tionto3-sulfocatechol[85,86,101](Fig.4).Thelatter
is a novel substrate for ring cleavage, and the spon-
taneous desulfonation is a consequence of the meta
ring cleavage enzyme, which is co-induced with the
ring-activating dioxygenase system.
The pathway thus contains a dioxygenation by a
putative class IB enzyme in EC 1.14.12.-, and the
reaction of this multi-component enzyme leads to
the spontaneous loss of ammonia (Fig. 4); if the
enzyme is supplied with TS, rapid dioxygenation
and desulfonation occur [53,85]. The desulfonation
of 3-sulfocatechol is caused by formation of the sul-
fo-aldehyde (Fig. 4), which is spontaneously hydro-
lyzed, probably distant from the enzyme active site
[85]. So this desulfonation is brought about by an
enzyme in EC 1.13.11.-, and it can equally well cata-
lyze a standard meta cleavage of catechol [85]. As
with the TS dioxygenase (desulfonating) in this or-
ganism (Section 6.3.2 and Fig. 3), the broad speci¢c-
ity of the enzymes is coupled to a very tight regula-
tion of enzyme induction [85].
The degradation of orthanilate is encoded on a
conjugative, IncP9 mega-plasmid, pSAH (172 kbp) Fig. 5. Convergence of catabolic pathways for arenesulfonates at
in Alcaligenes sp. strain O-1 [102,103]. Intra- and 4-sulfocatechol (4SC) and the ortho cleavage pathway established
interspecies matings to a plasmid-cured strain of Al- for that compound. None of the reactions leading to 4-sulfocate-
chol (from 2-(4-sulfophenyl)butyrate (SPB), sulfanilate (4AS) and
caligenes sp. O-1 and to Pseudomonas putida
benzene-1,3-disulfonate (BDS)) has been characterized [106 108],
PaW130, respectively, are possible. The transconju-
but the pathway in Hydrogenophaga palleronii S1 and Agrobacte-
gant of strain PaW130 acquires the ability to grow riumradiobacterS2hasbeenthoroughlyelucidated[106].
408
with orthanilate, indicating that all the required, fonate is required, because the fungus has already
pSAH-encoded genes were expressed. exported the non-speci¢c peroxidase.
6.3.6. The hydrolytic desulfonation of
p-aminobenzenesulfonate after ring cleavage 8. Aerobes assimilating sulfonate-sulfur
The degradative pathway for p-aminobenzenesul-
fonate illustrates yet another novel substrate for ring The bacterial utilization (assimilation) of organo-
cleavage, 4-sulfocatechol, that is subject to ortho sulfonates as sources of sulfur for growth di¡ers
cleavage during which the sulfonate is retained from the utilization of the same compounds as car-
[104] (Fig. 5). Formation of the lactone generates a bon sources in the enzymology involved and in the
relatively stable compound which includes a carbon genetic regulation of the enzymes. Whereas bacteria
atom carrying both an oxygen and a sulfono sub- usually organize catabolic enzymes in inducible op-
stituent; the previously stable C sulfonate bond is erons (see above), the utilization of sulfur is regu-
now subject to hydrolysis to sul¢te and maleylace- lated at the global level of the stimulon [116]. The
tate.ThepathwayoriginallyfoundinHydrogenopha- initialobservationinvolvedtwodimensionalgel-elec-
ga palleronii S1 and Agrobacterium radiobacter S2 trophoresis and showed that, under sulfate-starva-
[105,106], has also been found in a mixed culture tion conditions, E. coli and many other Gram-pos-
which degrades benzene-1,3-disulfonate [107] and itive and -negative bacteria each produces a set of
seems to be involved in the degradation of 2-(4-sul- proteins which is absent in the presence of sulfate.
fophenyl)butyrate[108].Thisnovelsubstrateforring These proteins were termed sulfate-starvation in-
cleavagethusful¢lstheroleonewouldpredictforit; duced (SSI) proteins [117] and some of them have
acting as a focal point for converging degradative been identi¢ed in E. coli (e.g. cysteine synthase
pathways. It will be interesting to see whether all CysK, sulfate-binding protein Ssp) [118] and in P.
intermediates of LAS degradation, and not just this aeruginosa (e.g. a sulfate-binding protein) [119].
sulfophenylbutyrate, follow this pathway (Fig. 5), The genes regulated by the SSI stimulon are ex-
which has not been postulated in any review (cited pressed when sulfate (and/or cysteine and perhaps
in [17]). Other sulfonates could also be channelled another, species-speci¢c compound) has been ex-
through 4-sulfocatechol, e.g. p-chlorosulfobenzoate hausted. The stimulon also seems to regulate the
[109], whereas m-amino- and m-nitrobenzenesulfo- distribution of sulfur within the cell [116].
nates [110,111] could theoretically be channelled The natural products taurine, cysteate and isethi-
through 3- or 4-sulfocatechol. The unidenti¢ed or- onateareusedassolesourcesofsulfurbyarangeof
ganism, strain S1, which Locher et al. [111] isolated bacteria and yeasts [120 124]. Where this has been
to utilize sulfanilate obviously also usedthe pathway examined at the molecular level, as yet only in E.
in Fig. 5, because 4-sulfocatechol was excreted tran- coli,itturnsoutthattheenzymologyistotallydi¡er-
siently. ent from the corresponding reaction when the car-
bonmoietyisutilized.Taurineisnolongersubjectto
transamination and hydrolysis (Section 6.1. and Fig.
7. Fungal peroxidases and arenesulfonates 6) but to a 2-oxoglutarate-dependent dioxygenation
(EC 1.14.11.-) that presumably yields the unstable
Desulfonations of aromatic sulfonates in fungi 1-hydroxy-2-aminoethanesulfonate which spontane-
have also been implied, because CO from the ring ously loses sul¢te with concomitant formation of
2
carryingthesulfonatewasreleased[112],orobserved aminoacetaldehyde. Taurine is the preferred sub-
directly [113]. These reactions have been attributed strate, but pentanesulfonate, hexanesulfonate, 3-(N-
to (extracellular) peroxidases [113,114], which some- morpholino)propanesulfonate (MOPS) and 1,3-di-
timesleadtoextensivedestructionofthecompounds oxo-2-isoindolineethanesulfonate were also desulfo-
involved,essentiallyas inthe enzymiccombustionof nated at signi¢cant rates; the ¢rst three sulfonates
lignin [115]. In these cases, no transport of the sul- also serve as sulfur sources for E. coli [55,125].