Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

24 Pages·2008·0.62 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,Sept.2008,p.471–494 Vol.72,No.3
1092-2172/08/$08.00(cid:1)0 doi:10.1128/MMBR.00008-08
Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.
Biosynthesis and Functions of Mycothiol, the Unique Protective
Thiol of Actinobacteria
Gerald L. Newton, Nancy Buchmeier, and Robert C. Fahey*
DepartmentofChemistryandBiochemistry,UniversityofCalifornia,SanDiego,LaJolla,California92093-0314
INTRODUCTION.......................................................................................................................................................471
STRUCTURE,PREPARATION,PROPERTIES,ANDDISTRIBUTIONOFMSH.........................................472
MSHBIOSYNTHESIS...............................................................................................................................................473
TheBiosyntheticPathway......................................................................................................................................473 D
o
MshA,theMSHGlycosyltransferase,andMshA2,theMSHPhosphatase...................................................473
w
MshB,theMSHDeacetylase.................................................................................................................................475 n
MshC,theMSHLigase.........................................................................................................................................476 lo
MshD,theMSHSynthase(MSHAcetyltransferase)........................................................................................477 a
d
RegulationofMSHBiosynthesis..........................................................................................................................477 e
MUTANTSINMSHBIOSYNTHESIS....................................................................................................................478 d

Mycobacteriumsmegmatis........................................................................................................................................478 fr
o
Poorgrowthduringinitialculture...................................................................................................................479 m
Sensitivitytoreactiveoxygenandnitrogenspeciesandredoxcyclingagents...........................................479
h
Sensitivitytoalkylatingagents.........................................................................................................................479 t
t
Resistancetoisoniazidandtoethionamide....................................................................................................479 p
:
Sensitivitytootherantibiotics..........................................................................................................................479 //
m
Productionofnovelthiols..................................................................................................................................479
m
Mycobacteriumtuberculosis.....................................................................................................................................480
b
MSHMutantsinOtherActinobacteria................................................................................................................480 r
.
MSH-DEPENDENTENZYMES...............................................................................................................................480 a
s
Overview...................................................................................................................................................................480 m
Mtr,theMSHDisulfideReductase(MycothioneReductase)..........................................................................481 .
o
Mca,theMSHS-ConjugateAmidase..................................................................................................................481 r
g
MscR,MSNOReductase/FormaldehydeDehydrogenase..................................................................................484 /

MaleylpyruvateIsomerase.....................................................................................................................................485 o
n
OtherEnzymes........................................................................................................................................................485
D
CELLULARTHIOLCONTENTANDREDOXSTATUS....................................................................................485
e
MSHTURNOVER......................................................................................................................................................486 c
MSHDRUGTARGETSINMYCOBACTERIUMTUBERCULOSIS....................................................................487 e
m
CONCLUSIONS.........................................................................................................................................................490
b
ACKNOWLEDGMENTS...........................................................................................................................................490 e
REFERENCES............................................................................................................................................................490 r
2
3
,

INTRODUCTION Actinobacteria is the production of mycothiol (MSH; also 2
0
designated AcCys-GlcN-Ins), a small thiol that is often 1
The class Actinobacteria is a very large and diverse group 8
of gram-positive, high-G(cid:1)C bacteria whose members pro- present in millimolar amounts and has analogous functions b
to glutathione (GSH), which is not found in Actinobacteria y
duce a variety of morphologies, from small cocci to highly
(84). g
branched mycelia (145). Members of the Actinobacteria are u
encountered in a wide range of ecosystems, from soil and In this review, we discuss what is currently known about es
seawater to the skin, lungs, and gastrointestinal tract of the biochemistry, biosynthesis, catabolism, and function of t
humans,andareresponsiblefortheproductionofcommer- MSH. The majority of the early work on MSH was carried
cialproducts,includingaminoacids,vitamins,andantibiot- out with mycobacteria, and this has laid the basis for more
ics (52, 155), as well as for causing important human dis- recent studies of other Actinobacteria. Several recent re-
eases, such as leprosy, tuberculosis, and diphtheria. In views which cover aspects of MSH biochemistry have ap-
addition, members of the Actinobacteria are used in the peared. An excellent review by Bhave et al. (6) considers
biodegradation of organic compounds during bioremedia- MSH metabolism in the larger general context of sulfur
tion (62). One feature which is common to most of the metabolism.DetailedcoverageofMSH-dependentenzymes
with bioinformatic analysis and comparison to analogous
GSH-dependentenzymesisfoundinareviewbyRawatand
*Correspondingauthor.Mailingaddress:DepartmentofChemistry
Av-Gay (114). Hand and Honek (41) consider MSH in the
and Biochemistry, University of California, San Diego, La Jolla, CA
context of the spectrum of thiols found in microorganisms.
92093-0314. Phone: [phone]. Fax: [phone]. E-mail:
[email protected]. Inthisreview,wehavesoughttominimizecoverageofthose
471
472 NEWTON ET AL. MICROBIOL.MOL.BIOL.REV.
suggestedthatmultipleconformationsareinrapidequilibrium
toproducethemeanresultdeterminedbyNMR.
AlimitingfactorinthestudyofMSHbiochemistryhasbeen
theavailabilityofMSHanditsprecursors.Oncethestructure
was established, its chemical synthesis was undertaken, but
generatingGlcN-Inswiththecorrectstereochemistryisachal-
lenging task. Jardine et al. (50) reported the production of a
mixture of GlcN-Ins isomers and use of a cell-free enzyme
preparationfromMycobacteriumsmegmatistoconvertthecor-
rect stereoisomer into MSH. An acetylated derivative of the
correct GlcN-Ins stereoisomer was synthesized by the Bewley
group(99),coupledtoAcCySmB,anddeacetylatedtogener-
ate the bimane derivative of MSH, MSmB. Lee and Rosazza D
(65)producedtheacetylatedGlcN-Insbyadifferentrouteand o
w
coupledittoN,S-diacetyl-L-cysteinetoproduceperacetylated n
MSH.BasichydrolysisgeneratedamixtureofMSHandMSH lo
disulfide(MSSM),withanoverallyieldof(cid:3)0.4%.Ahighyield ad
of GlcN-Ins was obtained in a recently reported synthesis of e
d
this substrate for use in a kinetic study of MshC (28). The
f
r
complexityandpooryieldsinvolvedinchemicalsynthesishave o
m
thus far made isolation of MSH a more efficient means of its

generation. h
FIG. 1. (A)StructureofMSH.(B)Conformationofmycothiol-S- In our laboratory, we isolate MSH from M. smegmatis by ttp
bimanedeterminedbyNMR.(Reprintedinpartwithpermissionfrom :
reference 69. Copyright 2003 American Chemical Society.) (C) Cys- using thiol affinity chromatography on activated thiopropyl //
m
teine complexed to heavy metal. (D) Transition state for S-N-acyl agarose, followed by elution with dithiothreitol and prepara-
m
migrationofS-acylcysteinethioesters. tive high-performance liquid chromatography (HPLC) of the
b
eluent(142).SteenkampandVogt(135)describedanalterna- r
.
a
tive approach to isolation of MSH involving reaction of the s
thiol with 2-S-(2(cid:4)-thiopyridyl)-6-hydroxynaphthyldisulfide to m
aspects that are well treated in these recent reviews and to .
generate 2-S-(mycothiolyl)-6-hydroxynaphthyldisulfide, fol- o
emphasize relevant chemical details where these have been r
lowedbysolid-phaseextractiononaC cartridgeandprepar- g
elucidated, but substantial overlap of coverage in some ar- 18 /
ative HPLC purification of the disulfide in the concentrated o
eas could not be avoided. eluent. The disulfide was reduced with dithiothreitol, and n

D
MSH was purified by preparative HPLC. GlcN-Ins can be
e
STRUCTURE,PREPARATION,PROPERTIES,AND generatedfromthebimanederivativeMSmBorotherS-con- c
e
DISTRIBUTIONOFMSH jugates of MSH (MSR) by treatment with the S-conjugate m
amidase Mca (see below) and purification by solid-phase ex- b
MSH, or 1-O-[2-[[(2R)-2-(acetylamino)-3-mercapto-1-oxopro- traction(86). e
r
pyl]amino]-2-deoxy-(cid:2)-D-glucopyranosyl]-D-myo-inositol, was ini- A key property of MSH is its resistance to autoxidation. 2
3
tially isolated and its structure elucidated from Streptomyces Cysteine undergoes heavy metal-catalyzed autoxidation much ,

strains(87,123)andfromMycobacteriumbovis(133).Thecom- morerapidlythanGSHdoes(45,138,140),apropertyattrib- 2
0
mon name mycothiol was proposed by Spies and Steenkamp utabletotheabilityofcysteinetoprovidethreemetalligands 1
8
(133).Thestructure(Fig.1A)involvesacysteineresidueinwhich inafavorablegeometricarrangement(Fig.1C).Theaffinities

the amino group is acetylated and the carboxyl group is amide ofthesubstituentsforheavymetalsdecreaseintheorder-S(cid:5)(cid:6) by
linked to D-glucosamine, which is in turn (cid:2)(1-1) linked to -NH2 (cid:6) -COO(cid:5). Since autoxidation produces hydrogen per- gu
myo-inositol.TheamidebondlinkingAcCystoGlcN-Insin oxide, which is lethal to cells (45), cysteine is a liability to e
MSHissufficientlyuniquethatitiscleavedonlybyspecial- aerobic cells and is generally maintained at a low concentra- s
t
izedenzymes,suchastheMSHS-conjugateamidase(Mca) tion.Cysteinederivativesfoundathighlevelsincellshavethe
(see below). aminoandcarboxylgroupsblockedinordertoslowautoxida-
Bewleyandcoworkers(69)determinedtheconformationof tion. In GSH, the (cid:7)-glutamyl and glycine residues reduce the
mycothiol-S-bimane (MSmB) in aqueous solution by nuclear Cu-catalyzedautoxidationrate8-to26-fold(45,138,140).In
magnetic resonance (NMR), producing the mean structure MSH, the acetyl and GlcN-Ins residues make Cu-catalyzed
shown in Fig. 1B. The conformation of the GlcN-Ins moiety autoxidationofMSHsome30-foldslowerthanthatofcysteine
was found to be essentially the same as that determined for and7-foldslowerthanthatofGSH(87).Cys-GlcN-Insautoxi-
GlcNAc-Ins, which is not surprising given the rigidity of the dizes(cid:3)11-foldfasterthanMSH,whichshowsthatblockingthe
GlcN and Ins rings. Theoretical calculations of the favored aminogroupofCysisofmajorimportance(94).WhyMSHis
conformation for MSH in an implicit water environment em- more resistant than GSH to autoxidation is not apparent but
ployed several different methods and generated several low- may be associated with the absence of any metal-chelating
energyclusters(40).Theglobalminimumclustermostclosely residuesinthemoleculeotherthanthethiolgroup.Themetal
matchedtheresultsdeterminedbyNMRonMSmB,anditwas ionautoxidationrateisinfluencedbythethiolpK andpossi-
a
VOL.72,2008 BIOSYNTHESIS AND FUNCTIONS OF MYCOTHIOL 473
TABLE 1. MSHcontentofActinobacteriaa MSH has been found to occur only in Actinobacteria , as
MSH summarized in Table 1 (64, 84, 89). However, not all Acti-
Strain(s)
contentb nobacteria produce MSH. One Arthrobacter species and one
eachofActinomycesandAgromycesspeciesgavenegativeanal-
ActinomadurahibiscaATCC53557................................................L
ActinomycesisraeliiATCC10049....................................................N yses from determinations for samples obtained at a single
AgromycesramosusATCC25173....................................................N growthtime.However,MSHlevelscanvarymarkedlywiththe
ArthrobacterglobiformisATCC8010..............................................N growth phase, and it would be imprudent to conclude that
CorynebacteriumdiphtheriaeUCSD21...........................................L
MSH is not produced by a species from analyses at a single
KocurearoseaATCC144.................................................................M
MarineStreptomycetaceaec growthtime.Thus,forthemarinestreptomyceteCNQ703,the
CNQ525,CNQ687,andCNQ695...............................................L valuesvaried40-foldoverthefullrangeofgrowthandsecond-
CNR252,CNQ698,CNQ719,andCNQ766..............................M arymetaboliteproduction(Table1).MSHhasnotbeenfound
CNR530andCNQ857..................................................................H
in representative samples of low-GC gram-positive bacteria,
CNQ703..........................................................................................L-M
MarineThermomonosporaceaec gram-negativebacteria,plants,fungi,oranimals(84).Thus,we D
CNR363..........................................................................................L concludethatMSHbiosynthesisisrestrictedtotheActinobac- ow
CNR431..........................................................................................H teria. n
MicrococcusagilisATCC966..........................................................L lo
MicrococcusluteusUCSD22...........................................................M a
MicrococcusluteusATCC4698.......................................................M MSHBIOSYNTHESIS de
MicrococcuskristinaeATCC27570.................................................L d
MicromonosporacarbonaceaATCC27144....................................L TheBiosyntheticPathway f
MicromonosporafloridensisJCM3265...........................................H ro
MicromonosporafulvopurpureaJCM5696.....................................M MSH biosynthesis was first elaborated in mycobacteria and m
MycobacteriumaviumNJH9151andNJH1854-4..........................M is accomplished in five steps, starting from 1L-myo-inositol-1- h
MycobacteriumchelonaeUCSD102...............................................M t
MycobacteriumfortuitumUCSD101..............................................M phosphate (1L-Ins-1-P or Ins-P), which is produced from glu- tp
Mycobacteriumsmegmatismc2155andmc26.................................H cose-6-phosphate (Glc-6-P) by inositol phosphate synthase :/
/
MycobacteriumtuberculosisATCC25618.......................................H (Ino1)(Fig.2).ThefirstdedicatedintermediateinMSHbio- m
MycobacteriumtuberculosisPZARUCSD100..............................M synthesis is 1-O-(2-acetamido-2-deoxy-(cid:2)-D-glucopyranosyl)-D- m
Nocardiasp.strainNRRL5646d....................................................M b
NocardiaasteroidesUCSD1andUCSD3....................................M myo-inositol-3-phosphate (GlcNAc-Ins-P), produced from r.
NocardiopsismutabilisATCC31520...............................................L UDP-GlcNAc and Ins-P by the glycosyltransferase MshA (91, as
SalinisporaarenicolaCNR107,CNR003,CNR643, 92).ThesecondstepinvolvesthedephosphorylationofGlcNAc- m
CNH725,andCNQ976c...............................................................M Ins-P, catalyzed by MshA2, which is encoded by a gene .o
StreptomycesclavuligerusATCC270674.........................................M thatisnotyetidentified.Thethirdstepisthedeacetylationof rg
SSttrreeppttoommyycceesscjuomeloicnojlionrenAsi3s(2A)T...C...C....2..9..8..6..4................................................................................MM 1-O-(2-acetamido-2-deoxy-(cid:2)-D-glucopyranosyl)-D-myo-inositol o/
StreptomyceslactamduransATCC27382.......................................M (GlcNAc-Ins) by the metalloprotein MshB (85) to yield 1-O- n
Streptomyceslividans1326................................................................M (2-amino-2-deoxy-(cid:2)-D-glucopyranosyl)-D-myo-inositol (GlcN- D
Ins),themajorintermediatefoundinextractsofmycobacteria e
aFromreference84,exceptasnoted. c
bN(none),(cid:8)0.05(cid:9)molpergdryweight;L(low),0.05to2(cid:9)molpergdry (14,93,118).Thesubstrateandproductsinvolvedinthefourth e
weight;M,(medium),2to10(cid:9)molpergdryweight;H(high),(cid:6)10(cid:9)molperg and fifth steps of MSH biosynthesis were first identified by m
dryweight. b
cFromreference89. Steenkampandcoworkers(9)andconfirmedbyAnderberget er
dFromreference64. al. (1). The fourth step in the pathway is the ATP-dependent 2
ligation of cysteine with GlcN-Ins by MshC, a homolog of 3
,
Cys-tRNA synthetase, to produce 1-O-[2-[[(2R)-2-amino-3- 2
blybytheredoxpotential,butthesehavenotbeenreported mercapto-1-oxopropyl]amino]-2-deoxy-(cid:2)-D-glucopyranosyl]-D- 01
for MSH. myo-inositol (Cys-GlcN-Ins) (125). The final step of MSH 8

Another problem for Cys in the cell comes from the pres- biosynthesis is acetylation of the amino group of cysteine in by
ence of coenzyme A (CoA) thioesters. S-Acyl transfer reac- Cys-GlcN-Insbyacetyl-CoAundercatalysisbyaGCN5acetyl- g
tionsbetweenthiolsandthioestersarefacilereactions(36,66), transferase,MshD,yieldingMSH(59). ue
and with the presence of acetyl-CoA and other acyl-CoAs at TheMSHbiosynthesisgenesforMycobacteriumtuberculosis, s
t
significant levels, the equilibrium generation of cysteine thio- Mycobacterium smegmatis, Streptomyces coelicolor, Corynebac-
ester species is highly likely. The difficulty is that cysteine teriumglutamicum,andRhodococcusjostiiRHA-1arelistedin
thioester species are able to undergo a facile intramolecular Table 2. All gene assignments for these organisms have been
acyl transfer reaction from sulfur to nitrogen via a favorable confirmed experimentally, with the exception of those for R.
cyclic transition state (Fig. 1D) to generate the more stable jostiiRHA-1.Ineachorganism,thegenesarenotarrangedin
amides, RCONH-Cys. With the amino group blocked, as in operonsandarefoundthroughouttherespectivegenome.
GSH or MSH, such rearrangements do not occur, and the
thioester can revert to the acyl-CoA if the concentration of
MshA,theMSHGlycosyltransferase,andMshA2,
CoA is favorable. This aspect of thiol biochemistry has re-
theMSHPhosphatase
ceivedlittleattention,inpartbecauseofthedifficultyinmea-
suring intracellular levels of specific acyl thioesters. The Cys MSHbiosynthesisbeginswiththeformationofthepseudo-
acyl transfer problem and the Cys autoxidation problem are disaccharide phosphate GlcNAc-Ins-P by MshA, a glycosyl-
keyreasonsthatcellularlevelsofCysarekeptlow. transferase identified by Tn5 mutagenesis of M. smegmatis
474 NEWTON ET AL. MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
FIG. 2. Biosynthesis of MSH. myo-Inositol-1-phosphate synthase (Ino1) generates Ins-P, MSH glycosyltransferase (MshA) links Ins-P to tp
GlcNAc,MSHphosphatase(MshA2)generatesGlcNAc-Ins,MSHdeacetylase(MshB)producesGlcN-Ins,MSHligase(MshC)linksCyswith :
/
GlcN-Ins,andMSHsynthase(MSHacetyltransferase;MshD)acetylatesCys-GlcN-InstoproduceMSH.Notethatmyo-inositolhasaplaneof /m
symmetrythroughC-2andC-5,makingC-1andC-3equivalentsoGlcN-Ins-Pcanbenamedasaderivativeof1L-Ins-1-Por1D-Ins-3-P,andthat m
bothsystemsareused. b
r
.
a
s
(91). MshA orthologs are found in the complete genomes of Streptomycescoelicolor,andNocardiafarcinicabutwassuccess- m
.
manyActinobacteria,andrepresentativeexamplesaregivenin fully produced by the Blanchard group from Corynebacterium o
r
Table 2. MshA is the only identified member of the GT-B glutamicum(146).TheenzymewasfoundtoexhibitMichaelis- g
/
superfamily (CAZy [http://www.cazy.org]) in M. tuberculosis Mentenkineticswithboth1L-Ins-1-PandUDP-GlcNAc,pro- o
that is not directly involved with cell wall biosynthesis (5). It ducingK valuesof240(cid:10)10and210(cid:10)20(cid:9)M,respectively, n
m
was initially found that M. smegmatis mshA mutants produce and a k value of 12.5 (cid:10) 0.2 s(cid:5)1. The rate expression with D
cat e
noGlcN-InsorGlcNAc-Ins(91,96),buttheactualsubstrates variationofbothsubstratescorrespondedtothatexpectedfor c
e
andbiochemistryofMshAwereonlyrecentlyelucidated(92). a sequential mechanism. Crystal structures were obtained for m
This was done by showing that extracts of M. smegmatis pro- the apo-enzyme, the binary complex with UDP, and the ter- b
duced GlcN-Ins from 1D,L-Ins-1-P and UDP-GlcNAc but not nary complex with UDP and 1L-Ins-1-P (146). The results er
frommyo-inositolor1-D-Ins-1-PandUDP-GlcNAc,indicating demonstrate that a substantial conformational change occurs 2
3
that 1L-Ins-1-P was the inositol donor for MshA. This was upon UDP binding, which generates the binding site for 1L- ,

verified with authentic 1L-Ins-1-P produced by Archaeoglobus Ins-1-P. Numerous residues involved in the binding of UDP 2
0
fulgidus inositol phosphate synthase (18). With 1L-Ins-1-P as and 1L-Ins-1-P could be identified, and a few of these are 1
8
thesubstrateofMshA,theproductispredictedtobeGlcNAc- showninFig.3.Thephosphateresidueofinositolisstabilized

b
Ins-P(Fig.2),andthiswasverifiedbyHPLC-massspectrom- byelectrostaticinteractionswithK78andR154andbymulti- y
etryanalysis(92). plehydrogenbonds,likelyexplainingwhytheenzymeisinac- g
u
The MshA protein proved difficult to clone and express in tivewithmyo-inositol(92).UDPisorientedandstabilizedby e
active form as a His -tagged protein from M. tuberculosis, interactionwithR231andK236aswellaswithotherresidues. s
6 t
TABLE 2. MSHbiosynthesisgenesfromselectedActinobacteriawithsequencedgenomes
Genenamein:
Gene
M.tuberculosis M.smegmatisa S.coelicolorb C.glutamicumc R.jostiid
mshA Rv0486 MSMEG0933 SCO4204 NCgl0389 ro02073
mshB Rv1170 MSMEG5129 SCO5126 NCgl1055 ro05935
mshC Rv2130c MSMEG4189 SCO1663 NCgl1457 ro00877
mshD Rv0819 MSMEG5854 SCO4151 NCgl2487 ro04861
aMycobacteriumsmegmatismc2155MSHbiosynthesispredictedproteinsare56%to76%identicaltotheM.tuberculosisH37Rvorthologs.
bStreptomycescoelicolorA3(2)MSHbiosynthesispredictedproteinsare42to55%identicaltotheM.tuberculosisorthologs.
cCorynebacteriumglutamicum(ATCC13032)MSHbiosynthesispredictedproteinsare35to56%identicaltotheM.tuberculosisorthologs.
dRhodococcusjostiiRHA-1MSHbiosynthesispredictedproteinsare47to66%identicaltotheM.tuberculosisorthologs.
VOL.72,2008 BIOSYNTHESIS AND FUNCTIONS OF MYCOTHIOL 475
Inositol is utilized in two known pathways in mycobacteria,
namely,theproductionofphosphatidylinositolforphosphati-
dylinositol mannosides and the biosynthesis of MSH. In M.
tuberculosis,inositolissuppliedas1L-Ins-1-Pfromglucose-6-P
(Fig.2)byinositolphosphatesynthase(Ino1)(81).Theessen-
tialino-1genewasknockedoutinM.tuberculosis,andmutants
were inositol auxotrophs, had normal levels of phosphatidyl-
inositolmannosides,lipomannan,andlipoarabinomannanbut
lowlevelsofMSH(Y.Av-Gay,personalcommunication),and
wereattenuatedforgrowthinSCIDmice(81).Thefindingof
anyMSHinthismutantissurprising.Theimmediateproduct
ofthereactionofino-1is1L-Ins-1-P,andtheknockoutmutant
requires myo-inositol to produce phosphatidylinositol for cell D
wallsynthesis.Thereareonlytwoknowntypesofenzymesthat o
w
utilize inositol-1-phosphate in mycobacteria, i.e., inositol n
monophosphatase (SuhB [101, 102] and CysQ [37, 43]) and lo
a
MshA. MshA cannot utilize myo-inositol or 1D-Ins-1-P, pro- d
duced from the action of phospholipase C activity on phos- e
d
phatidyinositol, to produce MSH (92). It is possible that a
f
FIG. 3. (A) PyMOL structure of selected active-site residues of myo-inositol kinase activity exists in M. tuberculosis and pro- ro
MshA from Corynebacterium glutamicum in complex with UDP and m
ducesthe1L-Ins-1-PrequiredforMSHbiosynthesis,aspostu-
Ins-P (146). (B) Summary of proposed catalytic mechanism for pro-
latedforM.smegmatis(92). h
ductionofGlcNAc-Ins-PbyMshA(146). t
t
p
:
/
MshB,theMSHDeacetylase /m
m
The highly conserved G22-G23 sequence is also part of the ThethirdMSHbiosynthesisenzyme,MshB,wasdiscovered
b
activesite,withG23hydrogenbondingtothe(cid:11)-phosphateof as a homolog of the MSH S-conjugate amidase (Mca) (see r
.
a
UDP. This explains why a G32D mutant of M. smegmatis below),anMSH-dependentdetoxificationprotein(85).MshB s
MshA (G32 is equivalent to G22 of C. glutamicum) is totally is a divalent metalloprotein which deacetylates GlcNAc-Ins m
.
inactive(91),sincereplacementofthehydrogensidechainof andalsohasoverlappingamidaseactivitywithMca(90).MshB o
r
glycine with the carboxymethyl side chain of aspartic acid is completely inactivated by the chelator 1,10-phenathroline, g
wouldbeexpectedtogreatlydisrupttheactivesite(Fig.3). and the apoenzyme is activated by Zn2(cid:1), Ni2(cid:1), Mn2(cid:1), and o/
Thecrystalstructuredataprovidedthebasisforamolecular Co2(cid:1)butnotbyCa2(cid:1)orMg2(cid:1).ThenativeformofMshBwas n

modeloftheternarycomplexofMshAwithUDP-GlcNAcand expected to be a Zn2(cid:1) metalloprotein, like its homolog Mca D
e
1L-Ins-1-P and for a proposed mechanism of catalysis (146). (see below), and this was confirmed by X-ray fluorescence c
e
The slow step of the reaction is considered to involve a nu- scanningandthecoordinationgeometrydeterminedbyX-ray m
cleophilic internal substitution in which the UDP-GlcNAc crystallography (71). A second structure of MshB was deter- b
bondionizestogenerateaC-1cationstabilizedbytheneigh- minedwithboundmercuryandoctylglycosideintheactivesite e
r
boringoxygenlonepair(Fig.3B).Simultaneously,the(cid:11)-phos- (73).Inbothcases,themetalbindingligandswereidentified 2
3
phate residue functions as a general base, with the hydroxyl asHis13,Asp16,andHis147,andAsp15wasidentifiedasa ,

groupofinositolallowingtheresultingoxygenaniontoattack plausible general base for deprotonation of water in the 2
0
the cationic site, generating a product with a retained config- catalytic step. 1
8
uration. A proposed catalytic mechanism for MshB, based on the

b
DephosphorylationofGlcNAc-Ins-PgeneratesGlcNAc-Ins, crystalstructure(71),isshowninFig.4.TheconservedAsp15 y
the substrate of MshB. This phosphatase, termed MshA2, is residueservesasageneralbaseforremovalofaprotonfrom g
u
currently unidentified. There are four inositol monophos- water, serving as a nucleophile in the attack on the amide e
phatase(IMP)homologstohumanIMPintheM.tuberculosis carbonylwhichispolarizedbythezinc.Thedevelopingnega- s
t
genome, including Rv2701c (suhB), Rv3137, Rv1604 (impA), tive charge on the carbonyl oxygen is stabilized by hydrogen
and Rv2131c (cysQ,) which are 28, 25, 23, and 19% identical, bondingtotheprotonatedHis144connectedinacharge-relay
respectively, to the human enzyme. M. tuberculosis SuhB has systemtoAsp146.Decompositionofthetetrahedralinterme-
beenclonedandexpressedandwasfoundtohaveactivitywith diaterequirestransferofaprotontotheamineoftheleaving
1D-Ins-P,Ins-2-P,glucitol-6-P,and2(cid:4)-AMP,similartothehu- GlcN-Ins. The general acid serving this function could be ei-
man, plant, and Escherichia coli IMP substrate specificity therprotonatedAsp15(71)orprotonatedHis144(46).
(102),butSuhBhasnotbeenassayedforMshA2activity.Since AstudyofthesubstratespecificityofM.tuberculosisMshB
no phosphatase mutants were identified in the screening of produced some curious results, as shown in Fig. 5 (90). Re-
chemical (96, 118) or transposon (59, 91, 118) M. smegmatis movaloftheInsresiduefromthenaturalsubstrate,GlcNAc-
mutant libraries for MSH-deficient mutants, it seems likely Ins,resultsina70-folddecreaseinrate,whereasreplacement
that more than one phosphatase in mycobacteria has MshA2 of the acetyl group by AcCys to generate MSH produces a
activity.Evenalkalinephosphatasehasbeenshowntodephos- 330-folddropinrate.MshB,likeMca(seebelow),hasamidase
phorylateGlcNAc-Ins-P(146). activity with MSR that in some cases (R (cid:12) CH COC H )
2 6 5
476 NEWTON ET AL. MICROBIOL.MOL.BIOL.REV.
synthetase,whichisunusualamongtRNAsynthetasesbecause
itisaZn2(cid:1)-containingprotein(83).
Cloning, expression, and purification of MshC from M. tu-
berculosisproveddifficultinE.colibyuseofconventionalHis
affinitytags.Thenativeenzymehasbeenclonedandexpressed
inanM.smegmatisMshC-deficientmutant,butitspurification
israthertedious(95).ExpressionandaffinitypurificationofM.
tuberculosis MshC have been achieved in an MshC M. smeg-
matismutantbyuseoftheN-terminalfusiontagsglutathione
S-transferaseandmaltosebindingproteinortheB1domainof
streptococcalproteinG(38).
MshC from M. smegmatis proved less fastidious, was ex-
FIG. 4. Proposed mechanism for the M. tuberculosis deacetylase pressed in E. coli as an N-terminally His-tagged protein, and D
o
MshB(71).TheactivesiteisformedwithAsp15,His13,andHis147 waspurifiedwithahighyield(28).TheM.smegmatisenzyme w
coodinatingaZnionrequiredforcatalysisandproteinstability.The wasusedforadetailedstudyofthereactionmechanismbythe n
active-siteZnalsopolarizestheacetylcarbon-oxygenbondofbound Blanchardgroup(28).Inordertostudythemechanismofthe lo
GlcNAc-Ins for attack by a hydroxyl ion generated from water by a
protonation of Asp15. The forming acetate is hydrogen bonded to MshC-catalyzed reaction, the group carried out a chemical d
e
His144orAsp15tocompletethehydrolysis. synthesis of GlcN-Ins, needed as a substrate, and conducted d
initialvelocityandinhibitionstudiesofthesteady-statemech- f
r
anism.Thedatawereconsistentwithabiuniunibipingpong o
m
approaches the activity with GlcNAc-Ins but is significantly mechanism,asshowninFig.6A,althoughtheorderofproduct
h
lower than the corresponding activity of Mca for most sub- release of AMP versus Cys-GlcN-Ins was not defined by the t
t
strates. An exception is CySmB-GlcN-Ins, which is 60-fold results. The rate-determining turnover of E-ATP-Cys to E- p
:
more reactive with MshB than with Mca and nearly 5-fold CysAdenylate-PPi competes with that of E-CysAdenylate- //m
morereactivewithMshBthanwithitsnativesubstrate,GlcNAc- GlcN-Ins to E-AMP-Cys-GlcN-Ins, with the rates being (cid:3)9.4 m
Ins. Since Cys-GlcN-Ins is present at only very low levels in and (cid:3)5.2 s(cid:5)1, respectively, yielding a calculated steady-state b
mycobacteria, it is unlikely that significant levels of S-conju- k of3.3s(cid:5)1,ingoodaccordwiththeexperimentalvalue(3.15 r.
cat a
gatesofCys-GlcN-Insareformedundernativeconditions.The s(cid:5)1). Steady-stateK valuesweredeterminedforATP(1.84(cid:10) s
m m
overlappingactivitiesofMshBandMcaareexaminedfurther 0.06 mM), Cys (0.10 (cid:10) 0.01 mM), and GlcN-Ins (0.16 (cid:10)
.
insubsequentsections. 0.05 mM). o
r
g
In a subsequent study, the initial steps of the MshC-cata-
/

lyzed reaction were examined by positional isotope exchange o
MshC,theMSHLigase n
using[(cid:11)(cid:7)-18O ]ATP(157).Inthepresenceofcysteine,forma-
6 D
ThefourthMSHbiosynthesisgeneproduct,MshC,formsan tion of E-CysAdenylate-PPi occurs with cleavage of the (cid:2)-P– e
amidebondbetweentheamineofGlcN-Insandthecarboxy- 16O bond to release PP with a single 16O and six 18O atoms. c
i e
late of L-cysteine. MshC was identified by purification of the Thereactionisreversibleandproducesisotopicmixingofthe m
ligaseactivityfromcrudeextractsofM.smegmatis,followedby 16O among the (cid:11)- and (cid:7)-phosphate oxygens of ATP, as mea- b
e
N-terminalsequencing(125).MshCisahomologofCys-tRNA sured by 31P NMR. The rate is dependent on the cysteine r

2
3
,

2
0
1
8

b
y

g
u
e
s
t
FIG. 5. Relative rates of M. tuberculosis MSH deacetylase (MshB) cleavage of amide bonds in acyl glucosamine derivatives. The substrate
specificityofMshBoverlapsthatofMSHS-conjugateamidase(Mca)(seeFig.11).ThenaturalsubstrateofMshBisGlcNAc-Ins,whoserateis
setat100asareference.
VOL.72,2008 BIOSYNTHESIS AND FUNCTIONS OF MYCOTHIOL 477
D
o
w
n
lo
a
FIG. 6. (A)BiuniunibipingpongkineticmechanismforM.smegmatisMshC(28).(B)StructuresoftheCysadenylateintermediateandits d
e
Cyssulfamoyladenylateinhibitoranalog. d

f
r
o
m
concentration,showingthattheformationofE-CysAdenylate- and is approximately twice the size of homologous proteins,

h
PP is reversible. However, no inhibition of exchange was ob- indicating that MshD is the result of a gene duplication (59).
i tt
served even at cysteine concentrations of 100 times the K Blanchard and coworkers (147) have determined a crystal p
m :
value, consistent with random binding of Cys and ATP (Fig. structure of M. tuberculosis MshD (Rv0819) showing that the //
m
6A) or ordered binding with Cys preceding ATP (Fig. 6A, C-terminaldomainbindsacetyl-CoAandthattheN-terminal
m
lowerroute).Therateofisotopicexchangewaseliminatedin acetyl-CoAbindingsiteisnotfunctional.Initialkineticstudies b
the presence of pyrophosphatase, showing that the PPi disso- indicated that acetyl-CoA is the preferred CoA thioester but r.a
ciates from the E-CysAdenylate complex and rebinds to pro- thatbothacetyl-CoAandpropionyl-CoAareMshDsubstrates s
m
ducethepositionalisotopicexchange.Inthepresenceofhigh (148). A crystal structure for the complex of MshD and CoA
.
concentrations of GlcN-Ins, the exchange activity was elimi- mixeddisulfidewithCys-GlcN-Insindicatesalargeconforma- o
r
nated. This result indicates that dissociation of PP from the tional change upon substrate binding (148). No inhibitors of g
i /
enzyme complex is faster than reformation of ATP and Cys MshDhavebeenreported. o
n
fromtheE-CysAdenylate-PP complex.Alternatively,ifGlcN- Kineticstudiesindicatedaternarycomplexmechanismcom-
i D
Ins can bind to this complex prior to dissociation of PP (an mon to the GNAT superfamily of N-acetyltransferases in
i e
alternativepathwaytothatshowninFig.6A),thenthisresult whichbothsubstratesmustbepresentforthereactiontooccur c
e
shows that reaction of the complex with GlcN-Ins is faster (148).Baseduponthecrystalstructureofthemixeddisulfide m
than the overall rate for regeneration and dissociation of ofCys-GlcN-InsandCoAandthepHdependenceofenzyme b
e
ATP from the enzyme. activity,thecatalyticmechanismshowninFig.7wasproposed.
r
The CysAdenylate analog 5(cid:4)-O-[N-(L-cysteinyl) sulfamoy- Glu234 is proposed to act as general base via an intervening 2
3
l]adenosine (Fig. 5B) is a known nanomolar inhibitor of Cys- water molecule, the Leu238 backbone NH stabilizes the neg- ,

and prolyl tRNA synthetases (53) and was shown to be a ativechargedevelopedinthetransitionstateonthecarbonyl 2
0
potentinhibitorofHis-taggedM.smegmatisMshC,havingan oftheacetylresidue,andTyr294providesaprotontogenerate 1
ATPcompetitiveinhibitionconstantof304(cid:10)40nM(28).Cys CoAinthethiolform(CoASH).TheKmvaluesforacetyl-CoA 8 b
sulfamoyladenylatewasalsofoundtobea50nMinhibitorof andCys-GlcN-Insweredeterminedtobe40(cid:10)5(cid:9)Mand82(cid:10) y
maltosebindingprotein-linkedM.tuberculosisMshC,whereas 22(cid:9)M,respectively. g
u
the aspartyl and seryl sulfamoyl adenosine analogs were less e
potentinhibitorsofM.tuberculosisMshC(38,38a),inaccord s
RegulationofMSHBiosynthesis t
withtheirexpectedloweraffinitiesfortheactive-sitezinc.
Currently, no crystal structure is available for MshC, but a There is a regulator preceding MshA in M. tuberculosis
crystal structure for the homologous E. coli Cys-tRNA syn- (Rv0485)annotatedasatranscriptionalregulatorsimilartoan
thasehasbeenreported(83). N-acetylglucosaminerepressorfromVibriocholerae.Similarly,
a transcriptional regulator precedes MshD in M. tuberculosis
(Rv0818), but no functional studies of the genes have been
MshD,theMSHSynthase(MSHAcetyltransferase)
reported.IndirectevidencesuggeststhatmRNAlevelsofthe
MSH synthase (MshD) is the fifth and final enzyme in the MSHbiosyntheticgenesarerelativelyconstantinM.tubercu-
MSHbiosynthesispathwayandcatalyzestheacetylationofthe losis,sincenoneofthesegeneshavebeenidentifiedinvarious
amino group of Cys-GlcN-Ins by acetyl-CoA (59). MshD is a studiesexaminingchangesinmRNAlevelsundervariouscon-
member of a large family of GCN5-related N-acetyltrans- ditions, such as stationary phase and low oxygen (151), expo-
ferases(GNATs)(23).Itappearstohavetwoseparateregions sure to hydrogen peroxide and palmitic acid, growth within
withsimilaritytothepfam00583domainforacetyltransferase macrophages (128), and exposure to nitric oxide (150). MSH
478 NEWTON ET AL. MICROBIOL.MOL.BIOL.REV.
and mca, and indirect expression of mshB, mshC, and mshD
(106). Treatment of cells with reagents that deplete MSH,
including the thiol oxidant diamide and the thiol alkylating
agents monobromobimane (mBBr) and N-ethylmaleimide,
leadstooxidationofRsrAandthereleaseof(cid:13)R,withsubse-
quent expression of the (cid:13)R regulon. The consensus promoter
sequence for mshA and mca was identified as GGAAT-N -
18
GTT, differing in only 1 nucleotide of spacer length from the
sequence determined by Paget et al. (104). Other genes that
undergo increased expression with (cid:13)R include rsrA and sigR,
producingamplificationoftheredoxsensingsystem,andtrxAB
(encoding thioredoxin and thioredoxin reductase), which also
provides reducing capacity. The sigR gene is absent in myco- D
bacteria,butarelatedgeneproduct,(cid:13)H(MT3320inM.tuber- o
w
culosisCDC1551),regulatesresponsestobothheatshockand n
redox stress (55). Several components of the thioredoxin sys- lo
tem were found to be regulated by (cid:13)H in M. tuberculosis, but ad
noneoftheMSHbiosyntheticgeneswerenoted. e
d

f
r
o
MUTANTSINMSHBIOSYNTHESIS
m

Mycobacteriumsmegmatis h
t
FIG. 7. Proposed catalytic mechanism for M. tuberculosis MshD tp
(148). The acetyl group from acetyl-CoA is transferred to the Cys Studies using mutants defective in MSH production have :/
amineofCys-GlcN-Insinaboundternarycomplex,withsubsequent beeninvaluableinidentifyingthegenesencodingeachofthe /m
release of CoASH, in a mechanism similar to that of other GCN5 MSHbiosyntheticenzymes,indeterminingwhichofthegenes m
acetyltransferases. is essential for the biosynthesis of MSH, and in defining the b
r
variousfunctionswithinthecellthatrequireMSH.Theinitial .a
studies on MSH biosynthesis were carried out with M. smeg- s
m
matis,andviablemutantswithreducedactivityofeachofthe .
biosynthetic enzymes are influenced by additional DNA se- o
MSH biosynthetic enzymes, except for MshA2, have been re- r
quences,sinceM.smegmatismutantslackingallMshCactivity g
covered.OneormoreoftheseM.smegmatisMSHmutantshas /
have been isolated containing lesions outside their structural exhibitedanincreasedsensitivitytoreactiveoxygenandnitro- o
gene(118). n
genspecies,toalkylatingagents,andtoantibiotics,suggesting D
SigmafactorsareinvolvedintheregulationofMSHmetab-
thatMSHisinvolvedinmutipledetoxificationmechanisms. e
olisminStreptomycescoelicolorA3(2).Asigmafactorrespon- c
sive to osmotic and peroxide stresses, (cid:13)B, has been linked to muAtasnutms imsagriyveonf tinheTtahbiolel c3o.nDteanttaofforthMe.Ms.msemgmegamtiastims cM21S5H5 em
upregulationofmshA,mshC,andmshDinS.coelicolor(63).S1 b
weretakenfrommultipleanalysesperformedinourlaboratory
nucleasemappingshowedthatexpressionofmshA,mshC,and e
(1, 59, 94). Mutants in the mshA gene produce undetectable r
mshDwasinducedbyosmoticstressandthattheinductionof 2
mofshMASaHndwmesrheCodnildynsolitgohctlcyurloinwear(cid:13)(B(cid:8)-d2e-fifocliden)timnutthaent(cid:13).LBenveullsl lMevsehlAs-odfeMficSieHntanmdutnaonnteisooflathteedMwSaHs ainctheremmeicdaialtmesu.tTanhte(fi9r6s)t 3, 2
thatcontainedasinglebasechangewithinmshA,producinga 0
mutant, suggesting that (cid:13)B may not be a major regulator of 1
G32D mutation, but contributions to the phenotype by addi- 8
MSHmetabolismundertheseconditions.

b
In S. coelicolor, the response to the production of disulfide y
bondswithinthecell(disulfidestress)isregulatedbythesigma g
factor (cid:13)R. The activity of (cid:13)R is controlled by the anti-sigma TABLE 3. ProducMtio.nsmoefgMmSaHtisamnudtaMntSsHintermediatesby ue
factor RsrA, which responds to changes in the intracellular s
Concn((cid:9)mol/gdryweight)ofMSH t
thiol-disulfideredoxbalance(54).Usingaconsensussequence
of (cid:13)R target promoters, Paget et al. (104) predicted that (cid:13)R Enzyme Strain orintermediate
GlcNAc-Ins GlcN-Ins Cys-GlcN-Ins MSH
regulates the expression of more than 30 genes, including
genes involved in thiol metabolism. When MSH levels were Wildtypea mc2155 (cid:8)0.1 0.2–1.0 (cid:3)0.008 10(cid:10)3
examined in a (cid:13)R-deficient mutant, they were fourfold lower MMsshhBAcb mMsyhcAo5::0T4n5 2.(cid:8)60(cid:10).001.2 (cid:8)(cid:8)00..0011 (cid:8)(cid:8)00..0012 1.(cid:8)00(cid:10).001.2
thanparentallevelsduringallphasesofgrowth.Morerecently, MshCd Tn1 NDe 2.6 (cid:8)0.002 (cid:8)0.004
theroleof(cid:13)RinMSHmetabolismwaselaboratedbyParkand MshDf mshD::Tn5 ND 0.35(cid:10)0.05 0.6–2 0.12(cid:10)0.01g
Roe(106).Acomplexisformedbetween(cid:13)Randthereduced aFromreferences1,59,and94.
form of the (cid:13)R regulator. Exposure of the oxidized disulfide bFromreference91.
cFromreference117andG.L.Newton,unpublisheddata.
formofRsrAtothiolsgeneratesRsrAred,andthiolsgenerated dFromreference118.
allowittobindZnandformRsrA(Zn)red(161).RsrA(Zn)red eND,notdetermined.
binds (cid:13)R, preventing it from activating its target genes. (cid:13)R fgFTrwoomtore3fe(cid:9)remnocle/gso59ffaonrmdy9l4-C.ys-GlcN-Insand0.12(cid:10)0.01(cid:9)mol/gofsuccinyl-
activatesdirectexpressionofahostofgenes,includingmshA Cys-GlcN-Inswerealsoproduced.
VOL.72,2008 BIOSYNTHESIS AND FUNCTIONS OF MYCOTHIOL 479
tionalchangesinthechemicalmutantwerenotexcluded(91). peroxide,menadione,plumbagin,andt-butylhydrogenperox-
Usingsite-directedmutagenesis,wereconstructedthismutant ide(withMshA,MshB,MshC,andMshDmutants)(116–118).
and confirmed that the single base change generating the TheMshAchemicalmutantandMshC-deficientmutants(Tn1
G32DmutantresultsinacompletelossinsynthesisofGlcNAc- and Tn2) were at least 10-fold more sensitive to killing by
Ins, GlcN-Ins, and MSH. In addition, an mshA::Tn5 mutant hydrogen peroxide in a 2-h broth exposure (96, 118). The
wasidentifiedfromaTn5transposonlibraryafterselectionfor MshAmutantwasalsoreportedtobemoresensitivetokilling
resistancetoisoniazid(100(cid:9)g/ml),acharacteristicwhichhad bygaseousnitricoxide(79).Whenbacteriawerecontinuously
previouslybeenestablishedasaphenotypeforMSH-deficient exposed to gaseous nitric oxide, toxicity to the mutant began
M.smegmatis(96,118).ThemshA::Tn5mutantwasdevoidof after4hofexposuretothegas,comparedto7hforwild-type
GlcNAc-Ins,GlcN-Ins,andMSH. M.smegmatis.
TheMshBmutantMyco504containsakanamycinresistance Sensitivitytoalkylatingagents.TheMSHS-conjugateami-
cassette at base 207 within mshB (117). This mutant accumu- dase(Mca)takespartinthedetoxificationofalkylatingagents,
lateselevatedlevelsoftheintermediateGlcNAc-Ins(Table3), suggestingthatMSH-deficientmutantsshouldbemoresensi- D
confirmingthatthemajordeacetylaseactivitywithintheMSH tive to these agents (86). The MshC mutants were two- to ow
biosynthetic pathway has been inactivated. The mshB mutant fourfold more sensitive to killing by the alkylating agents n
alsoproducesabout10%ofnormallevelsofMSH,indicating mBBr,iodoacetamide,and1-chloro-2,4-dinitrobenzene(118). lo
a
that the cell contains an additional activity that is capable of In a separate study using a disk diffusion assay, the MshA-, d
deacetylating GlcNAc-Ins. A possible candidate for this e
MshB-,MshC-,andMshD-deficientmutantswereallsensitive d
deacetylaseistherelatedproteinMca,whichhasweakGlcNAc- to1-chloro-2,4-dinitrobenzeneat0.2(cid:9)mol,andallexceptthe f
r
Ins deacetylase activity (136). However, complementation of MshB mutant were sensitive to iodoacetamide at 0.05 (cid:9)mol o
m
Myco504 with an expression plasmid expressing Mca did not
(116).
increasethelevelofMSHinthecomplementedmshBmutant h
Resistancetoisoniazidandtoethionamide.Oneofthemost t
(117),soinvolvementofanotherdeacetylaseispossible. tp
pronounced characteristics observed in the first MSH mutant :
TheTn1transposonmutantiscompletelydefectiveinMshC isolated was its (cid:6)25-fold increase in resistance to isoniazid //m
activity,hasundectablelevelsofCys-GlcN-InsandMSH,and
(96). In the subsequent isolation of additional MSH mutants, m
haselevatedlevelsofGlcN-Ins(118).Interestingly,thetrans- resistanceto100(cid:9)g/mlisoniazidwasusedintheinitialmutant b
poson insertion in this mutant and a companion MshC-defi- r
screen.Inthismanner,themshA::Tn5andmshD::Tn5mutants .a
cientmutant,theTn2mutant,isnotwithinthemshCgenebut were isolated (59, 91). The MshB-deficient mutant, which s
atanunknownlocationwithinthechromosome. m
makes5to10%ofnormalMSHlevels,hasbeenreportedtobe .
The mshD::Tn5 mutant is a transposon mutant which was o
onlyslightlymoreresistanttoisoniazidthanthewildtypeina r
alsoinitiallyidentifiedbyitsresistancetoisoniazid.TheMshD g
diskassay(116,117),suggestingthatmoderatelevelsofMSH /
mutantproducessmallamountsofMSHaswellashighlevels o
of the MshD substrate Cys-GlcN-Ins, a novel thiol (formyl- are sufficient to confer normal sensitivity to isoniazid. En- n
hanced resistance to ethionamide is another characteristic of D
Cys-GlcN-Ins),andmoderateamountsofanothernovelthiol,
the MshA-, MshB-, and MshD-deficient mutants, with com- e
succinyl-Cys-GlcN-Ins(59,94).Formyl-Cys-GlcN-Insisclosely c
pleteresistanceat50(cid:9)g/mlinadiskassay(116).Usinganagar e
related to MSH in structure, with the CH3CO- residue re- plate assay, the MIC of ethionamide for the mshB mutant m
placed by HCO- on the nitrogen of Cys. Experimentally, 99 b
formyl-Cys-GlcN-InshasbeenshowntoreplaceMSHinreac- Myco504was150(cid:9)g/ml,whichwassixfoldhigherthanthatfor e
tions of two MSH-dependent enzymes, Mca and Mtr (myco- theparentalstrain(117).Bothisoniazidandethionamideare r 2
prodrugswhichneedtobeactivatedintracellularlyandwhose 3
thionereductase),withreducedbutsignificantrates(94).This ,
suggeststhathighlevelsofformyl-Cys-GlcN-Insmaypartially reactivegroupsmustbeunmaskedbeforeinhibitingtheirtar- 2
gets(4).ThehighlevelsofresistanceoftheMSHmutantsto 0
substitute for MSH in the MshD-deficient mutant. Chemical 1
isoniazidandtoethionamidesuggestthatMSHisinvolvedin 8
transacylation of Cys-GlcN-Ins by acetyl-CoA and succinyl-

CoA is the most likely source of the small amounts of MSH theactivationofthesedrugs. by
andsuccinyl-Cys-GlcN-Insproducedinthismutant. Sensitivitytootherantibiotics.Incontrasttotheenhanced g
The following is a summary of phenotypic characteristics resistancetoisoniazidandtoethionamide,theMSHmutants ue
observedamongthevariousM.smegmatisMSHmutants. havedisplayedincreasedsensitivitiestoavarietyofantibiotics, s
t
Poor growth during initial culture. We have observed that including rifamycin, streptomycin, erythromycin, and azithro-
MshA- and MshC-deficient mutants are slower to grow from mycin (96, 116–118). For example, the MshA mutant was 10-
frozenstocksduringtheinitialstagesofculture,andthismay foldmoresensitivetorifampinand(cid:3)20-foldmoresensitiveto
reflecttheirlackofMSH.ThissuggeststhatMSHmayprovide erythromycinthanthewildtypebyuseofEteststrips(116).
someprotectionduringadaptationtogrowthinanoxygen-rich Productionofnovelthiols.ThemshDmutantproducestwo
environmentandduringtheinitiationofcellularmetabolism. novel thiols, formyl-Cys-GlcN-Ins and succinyl-Cys-GlcN-Ins
Sensitivity to reactive oxygen and nitrogen species and re- (94). The blockage at the MshD step results in an accumula-
dox cycling agents. The MSH mutants have been reported to tion of high levels of Cys-GlcN-Ins within the cell. It seems
exhibit some degree of sensitivity to reactive oxygen species likely that cellular succinyl-CoA undergoes S-transacylation
andredoxcyclingagents,withtheresultsdependentuponthe withCys-GlcN-Ins,followedbyS-Nacyltransfer,toformsuc-
typeandamountofcompoundusedandthemannerinwhich cinyl-Cys-GlcN-Ins(Fig.1D),aprocessshowntooccurchem-
theassaywasperformed.Compoundswhichproducedgreater ically in solution (94). This is similar to the nonenzymatic
inhibitionofgrowthinadiskdiffusionassayincludedhydrogen formationofMSHviaacetyl-CoAinthemutant.Theoriginof
480 NEWTON ET AL. MICROBIOL.MOL.BIOL.REV.
TABLE 4. ProductionofMSHandMSHintermediatesby growthinmacrophages,anmshDmutantwasoneofthemost
M.tuberculosismutants impaired(120).ThemshDmutantfailedtogrowinculturesof
Concn(nmol/109cells)ofMSHorintermediate primary murine macrophages that modeled all stages of the
host immune response, including inactivated macrophages,
Strain GlcNAc-Ins GlcN-Ins Cys-GlcN- exMpoSnHenitnial- stMatSioHnairny- gamma interferon-preactivated macrophages, and gamma in-
Ins
phasecells phasecells terferon-postactivated macrophages. The mshD mutant had
M.tuberculosis 0.7–3 4–10 (cid:8)0.1 10–20 30–40 notpreviouslybeenidentifiedasbeingdefectiveforgrowthin
Erdmana the spleens of mice after intravenous infection of mice (127),
mshBmutantb 30–200 0.2–3 (cid:8)0.2 2–3 50–60
indicating that the mshD gene is critical for survival under
mshDmutantc NDf NDf 3–11 0.1–0.3d 0.7–2.2e
distinct conditions. These results indicate that growth of M.
aFromreferences13and14. tuberculosis in environments where antimicrobial factors, in-
bFromreference14.
cFromreference13. cluding reactive oxygen and reactive nitrogen intermediates,
dThreeto5nmol/109cellsofformyl-Cys-GlcN-Inswasalsoproduced. are formed, such as within macrophages, relies upon MSH- D
eSevento17nmol/109cellsofformyl-Cys-GlcN-Inswasalsoproduced. dependentsystems. o
fND,notdetermined. w
n
lo
MSHMutantsinOtherActinobacteria a
formyl-Cys-GlcN-InsinthemshDmutantislikelytobeenzy- d
e
matic. MutantsinMSHbiosynthesishavebeenisolatedinseveral d
otherActinobacteria,andthosemutantswhichhavebeenchar- f
r
Mycobacteriumtuberculosis acterized have phenotypes that are quite similar to those ob- om
served with the M. smegmatis mutants. In Streptomyces coeli-

Usingatemperature-sensitiveshuttlephasmidtointroduce color,disruptionofthemshA,mshC,ormshDgeneabolished ht
targeteddisruptionswithinthechromosomeofM.tuberculosis all detectable MSH, while a MshB mutant produced 10% of tp
:
Erdman,wehavebeenabletogenerateviablemutantsinthe wild-type levels (107). This indicates that the deacetylase ac- //
m
mshB(14)andmshD(13)genesbutnotinthemshA(12)and tivityoftheMshBproteincanbeprovidedbyotherenzymes, m
mshC(124)genes(Table4).ThemshBmutant,whichcarries whileMshA,MshC,andMshDareessentialforMSHbiosyn- b
a disruption in gene Rv1170, produced (cid:3)20% of wild-type thesis in S. coelicolor, which is identical to the situation in M. r.
a
levels of MSH during exponential growth. With prolonged smegmatis. The MshA and MshC mutants were reported to s
culture, MSH levels increased over 20-fold, to levels signifi- differentiatemoreslowly,butallofthemutantsgrewaswellas m
.
cantly higher than those in wild-type M. tuberculosis (14). the wild type in yeast extract-malt extract liquid medium. o
r
GlcNAc-Ins levels were dramatically elevated and GlcN-Ins CorynebacteriumglutamicumisanMSH-containingsoilorgan- g
/
levelswerereducedinthemutant,establishingthattheRv1170 ism that is commercially used to produce amino acids and o
geneencodesthemajorMshBactivityintheMSHbiosynthetic vitaminsandthatcanmetabolizearomaticcompounds.Muta- n
D
pathwayofM.tuberculosisbutthatanotherdeacetylaseactivity tions in mshC and in mshD resulted in no MSH, while a
e
can partially substitute for Rv1170 activity during MSH bio- mutation in mshB reduced MSH levels (29). The mshC and c
synthesis. The mshD mutant produced (cid:3)1% of normal MSH mshDmutantswereunabletogrowinmediawithgentisateor em
levels and high levels of the MshD substrate Cys-GlcN-Ins 3-hydroxybenzoateasacarbonsource,andthisobservationled b
(13).ThedominantthiolintheMshDmutantwasformyl-Cys- tothediscoveryofanMSH-dependentmaleylpyruvateisomer- er
GlcN-Ins,similartothesituationinM.smegmatis. ase. A MshD mutant has been constructed in Amycolatopsis 2
3
Because the MshB- and MshD-deficient mutants produce mediterranei,astreptomycetestrainusedtoproducerifamycin ,

significantlevelsofeitherMSHortheMSH-likethiolformyl- SV (17). Although MSH was not quantitated in this mutant, 20
Cys-GlcN-Ins,theyarenotidealorganismsforcharacterizinga themutantwasreportedtogrowslowerthanthewildtypeon 1
8
MSH-freestateinM.tuberculosis.However,certaincharacter- plates and was more sensitive to hydrogen peroxide and the
b
isticswhichsuggestfunctionsforMSHinM.tuberculosisdur- antibioticsapramycinanderythromycin. y
ingitsgrowthandresponsetostresswereapparentinbothof g
u
these mutants. Both the MshB- and MshD-deficient mutants e
grew poorly on agar media lacking Middlebrook OADC sup- MSH-DEPENDENTENZYMES s
t
plement (oleic acid, NaCl, albumin, dextrose, and catalase),
Overview
resultinginareducedplatingefficiencyandsmallercolonysize.
ThedefectwasmostpronouncedfortheMshD-deficientmu- Enzymes which require MSH are involved in a number of
tant,witha(cid:6)3-logdropinplatingefficiencyonplateslacking cellular processes, including detoxification of electrophilic
OADC versus ADS supplement (albumin, dextrose, NaCl) compounds, inactivation of reactive oxygen and nitrogen spe-
(13). Log-phase cultures of the MshB-deficient mutant were cies,reductions,andisomerizations.Theactivitiesofsomeof
(cid:3)2.5 times more sensitive to the toxic oxidant cumene hy- themorewell-characterizedenzymesaresummarizedinFig.8.
droperoxide afte r 7 h ofexposure and 2 to 4 times more MSH reacts spontaneously with formaldehyde to produce an
sensitive to rifampin, depending upon the concentration of adduct, H C(OH)SM, which is a substrate for the formalde-
2
drug. The MshD-deficient mutant was moderately more sen- hydedehydrogenaseAdhE2(103).AdhE2-catalyzedoxidation
sitive to killing by hydrogen peroxide and more restricted in offormaldehydetoformatedetoxifiesformaldehydeoriginat-
growth in moderately acidic (pH 5.5) medium. In a genome- ing from metabolic or environmental sources. It was later
widescreenofnonessentialM.tuberculosisgenesrequiredfor shownthatthemycobacterialenzymeexhibitsS-nitrosomyco-