Research Article Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic ...

14 Pages·2015·3.38 MB·English

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BioMedResearchInternational
Volume2013,ArticleID458571,13pages
http://dx.doi.org/10.1155/2013/458571
Research Article
Structure-Based Mechanism for Early PLP-Mediated Steps of
Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction
MartinoL.DiSalvo,1J.NeelScarsdale,2GalinaKazanina,2RobertoContestabile,1
VerneSchirch,3andH.TonieWright3
1DipartimentodiScienzeBiochimiche,SapienzaUniversita`diRoma,00185Roma,Italy
2CenterfortheStudyofBiologicalComplexityandInstituteforStructuralBiologyandDrugDiscovery,Richmond,
VA23284-2030,USA
3DepartmentofBiochemistryandInstituteofStructuralBiologyandDrugDiscovery,VirginiaCommonwealthUniversity,
Richmond,VA23219,USA
CorrespondenceshouldbeaddressedtoMartinoL.DiSalvo;[email protected]
Received6June2013;Accepted26June2013
AcademicEditor:AlessandroPaiardini
Copyright©2013MartinoL.DiSalvoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
SerinehydroxymethyltransferasecatalyzesthereversibleinterconversionofL-serineandglycinewithtransferofone-carbongroups
toandfromtetrahydrofolate.ActivesiteresidueThr254isknowntobeinvolvedinthetransaldiminationreaction,acrucialstep
󸀠
inthecatalyticmechanismofallpyridoxal5-phosphate-(PLP-)dependentenzymes,whichdeterminesbindingofsubstratesand
releaseofproducts.InordertobetterunderstandtheroleofThr254,wehaveexpressed,characterized,anddeterminedthecrystal
structuresofrabbitcytosolicserinehydroxymethyltransferaseT254AandT254Cmutantforms,intheabsenceandpresenceof
substrates.Thesemutantsaccumulateakineticallystablegem-diamineintermediate,andtheircrystalstructuresshowdifferences
intheactivesitewithrespecttowildtype.Thekineticandcrystallographicdataacquiredwithmutantenzymespermitustoinfer
thatconversionofgem-diaminetoexternalaldimineissignificantlyslowedbecauseintermediatesaretrappedintoananomalous
positionbyamisorientationofthePLPring,andanewenergybarrierhampersthetransaldiminationreaction.Thisbarrierlikely
arisesfromthelossofthestabilizinghydrogenbondbetweenthehydroxymethylgroupofThr254andthe𝜀-aminogroupofactive
siteLys257,whichstabilizestheexternalaldimineintermediateinwildtypeSHMTs.
1.Introduction enzymes exist in complexes that absorbs in the 310nm to
500nmrangeastheresultofaconjugated𝜋-electronsystem.
Serine hydroxymethyltransferase (SHMT: EC 2.1.2.1) is These absorption properties have played an important role
󸀠
a ubiquitous pyridoxal 5 -phosphate- (PLP-) dependent inelucidatingthemechanismsofPLPadditionandcatalysis,
enzyme that catalyzes the reversible interconversion of L- since several intermediates on the reaction pathway have
serineandglycine,coupledtotheformationandbreakdown unique structural and absorbance characteristics [10, 11].
of 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu) [1, SHMT is distinctive among the PLP-dependent enzymes
2]. Because of its essential role in one-carbon units in the number of these absorbing complexes that can be
metabolism, SHMT has been often indicated as a potential observed, and these have been exploited to determinate
target of chemotherapeutic agents [3–5]. It also catalyzes kinetic rates for their interconversion by stopped-flow and
the conversion of 5,10-methenylene- to 5-formyl-H4PteGlu temperaturejumpspectroscopy[11–13].
󸀠
[6], the transamination and racemization of D- and L- In PLP-dependent enzymes, the 4 -aldehyde of PLP is
alanine [7], the retro-aldol cleavage of erythro and threo boundasanaldiminetothe𝜀-aminomoietyofanactivesite
isomers of both L-threonine and L-𝛽-phenylserine [8], and Lysresidueinwhatiscalledthe“internalaldimine.”Forthose
the decarboxylation of aminomalonate [9]. PLP-dependent PLP-dependent enzymes that catalyze reactions involving
2 BioMedResearchInternational
Enz
Lys257
N
HC H
2−(Oen3PoOlimiCnIHne t2feorrnmal, aAlHNdmi+amxi≈ne3 O 40nm2−)O3POHCH2C:ONOHH−C2R NL+yOsH25−7 Enz 2−O3PHOOHCCHHO−2NO−+RHCH N¨LysOH25−7Enz 2−O3PHO CCHO:2NOH−RHHC NLy+sO2H57−Enz 2−O3PO CH2 HHC NC+OOHO−R−Lys257Enz
HO NLys257 Enz (ketoenamiInneHtNe fr+onraml ,aAldmimaxin≈e 430nm) (AGemmaxNH-d≈+ia3m43innem I) (AGmemax-d≈NHia3+m43innem II) E(AxtmeraNHnxa+≈l a4l2d6imnmin)e
HC H
2−O3PO CH2 O− COO− R
(caHrybdinraotleadm iinnHNtee,+rAnaml aaxld≈im34in0enm) 2−O3PO CH2 HC N+HO−
N
H
Quinonoid intermediate
(Amax≈498nm)
Scheme1:MechanismoftransaldiminationreactionofSHMT.
substrateaminoacids,theinitialstepinthecatalyticreaction only the T226A mutant of ecSHMT (Thr226 in ecSHMT
is the formation of the “geminal diamine” (gem-diamine) numberingisequivalenttorcSHMTThr254)hadsignificant
󸀠
between the C4 aldehyde of PLP and the amino group of spectralandkineticdifferencesfromthewildtypeenzyme.
the substrate. The orientation of the substrate in the active Therewasa32-foldlower𝑘 intheconversionofL-serineto
cat
sitewithrespecttotheplaneofPLP,towhichitiscovalently glycine,andtheT226Amutantwasvirtuallyinactivetoward
linked through its amino group, in turn determines which cleavageofL-allo-threoninecomparedtowildtypeecSHMT.
ofthethreesubstituentbondsonC𝛼ofthesubstratewillbe Furthermore,inthepresenceofL-serinetheT226Amutant
cleaved.In1966,Dunathan[14]providedaunifyingconcept exhibitedalargespectralabsorbancepeakat343nm,which
forthespecificselectionofthesubstratescissilebondinPLP- is characteristic of a gem-diamine intermediate and only a
dependent enzymes, which has been confirmed in solution smallpeakat425nmcharacteristicoftheexternalaldimine.
andstructuralstudies.Inhisproposal,allbondsbrokenand Stopped-flow analysis showed that the 343nm peak was
madeonthecatalyticpathwayarethenearestperpendicular formed rapidly, but its conversion to the 425nm absorbing
totheconjugated𝜋systemofthePLPring.Theresultingcar- peak was slow [17]. Since the gem-diamine is generally a
banionatC𝛼ofthesubstrateaminoacidisstabilizedbyreso- short-lived intermediate on the reaction pathways of PLP-
nancewiththe𝜋-electronsysteminthepyridineringofPLP. dependentenzymes,thisT226AecSHMTmutantofferedthe
Thisintermediateonthecatalyticpathwayisreferredtoasthe opportunitytoinvestigatethestructuralchangesthatappar-
“quinonoid” complex and absorbs near 500nm (Scheme1). entlyslowedtheconversionofthegem-diamineintermediate
Solution studies have shown that SHMT passes through to the external aldimine. We were unable to obtain crystals
severalordered,spectrophotometricallyidentifiableinterme- for the T226A mutant of ecSHMT. However, we were able
diatesthatreflectchangesintheelectronsystemofthePLP tocrystallizethehomologousT254AandT254Cmutantsof
cofactor[12,15]andareconsistentwithDunathan’sproposal. rcSHMT. We report here the kinetic properties and crystal
Prior to the availability of crystallographic structure structuresoftheT254mutantsofrcSHMTandtheirglycine
information on SHMT, it was noted that the amino acid and L-serine complexes. In addition, we have increased the
sequencesofSHMTsfromdiversespecieshaveaconserved resolutionofthestructureofwildtypercSHMTto2.1A˚ asan
runof4threonineresiduesterminating2residuesupstream aidinexaminingdetailsofthegem-diaminestructures.
254 257
of the active site lysine: V-V-T-T-T -T-H-K (PLP)-T
(numberingisthatforrabbitcytosolicserinehydroxymethyl- 2.ResultsandDiscussion
transferase(rcSHMT))[16].Todeterminethepossibleroles
ofthisconservedsequenceinSHMTcatalysis,eachthreonine 2.1. Spectroscopic Studies. Wild type rcSHMT exhibits a
of this active site stretch was mutated in ecSHMT to an characteristicmajorsingleabsorptionbandwithmaximum
alanine, and the effects of the changes on the spectral and intensityat430nm,duetotheprotonatedinternalaldimine
kinetic properties were investigated [17]. It was found that betweenenzymeandcofactor.Also,aminorbandisobserved
BioMedResearchInternational 3
at340nm(Scheme1).Theadditionofsaturatingconcentra- for the Thr to Ala mutant and 531-fold for the Thr to Cys
tionsofglycineresultsinadecreaseofthemajorabsorption mutant.Similarly,𝐾𝑚valuesforL-allo-threoninewerefound
band,withaslightblue shiftat426nmandtheappearance tobeonlyslightlyhigherthanwildtype(lessthan2-foldfor
of a well-defined band at 343nm. A small band centered both mutants), whereas 𝑘 values showed a 9- to 29-fold
cat
at 498nm is also visible. It is well established that these decreaseforT254AandT254Cmutants,respectively.
absorptionbandscorrespondtotheformationoftheexternal TobetterunderstandtheroleofThr254intheformation
aldimine,thegem-diamine,andthequinonoidintermediates, and breakdown of the gem-diamine intermediate and thus
respectively[1,12,18].Allabsorptionbandsofwildtypeand on the transaldimination reaction, the rate of the spectral
mutantenzymesareshowninFigure1.
changesoccurringwhenL-serineandglycinewereaddedto
Like the wild type, the T254A mutant form exhibits
theenzymewasdeterminedatdifferentpHvaluesbymeans
a 430nm absorption band (although much less intense of stopped-flow measurements. The 𝑘on and 𝑘off values for
than wild type), indicative of the presence of an internal
therateofformationandbreakdownoftheenzyme-substrate
aldimine, and also shows an important band at around
complex absorbing at 343nm were determined from a plot
340nmthatmayrepresenteithertheenolimineformorthe of𝑘 versussubstrateconcentration,afterlinearregression
carbinolamine, a hydrated form of the internal aldimine in obs
of data and extrapolation of the slope and intercept values
whichthewatermoleculemimicsthesubstratenucleophilic
(Table2).ForbothmutantsandforbothsubstratesateachpH
attackandthegem-diamineformation.AsshowninFigure1, value,𝑘 wasalinearfunctionofsubstrateconcentration,as
this band is usually present in very low concentration in obs
expectedforasecond-orderreaction.Thesecond-orderrate
the freshly purified wild type enzyme. The addition of
constantsforsubstrateadditionandgem-diamineformation
a saturating concentration of glycine to T254A mutant (𝑘on)werecorrectedfortheconcentrationoftheaminoacid
resultsinthealmostcompletelossofabsorbanceat430nm, anionic form at each pH value and are listed as 𝑘󸀠 . The
with a concomitant increase of absorbance at 343nm. This on
anionic form of the 𝛼-amino group is assumed to be the
effect was also observed with E. coli SHMT T226A mutant
truesubstrateforthetransaldiminationreaction.InthepH
(residue Thr226 of ecSHMT corresponds to residue Thr254
range used in the experiments (6.4–8.0), the concentration
inrcSHMT).Kineticstudiesonthelattermutantshowedthat
of the anionic form of the amino acid substrates increases
thecomplexabsorbingat343nmisformedinabimolecular
step providing strong evidence that it is indeed the gem- afrboomut34.30t⋅i1m0e4st.oTh1e0.𝑘7on⋅1v0a4luse−s1⋅iMnc−r1eawshedenwLit-hseprHineanwdasvaursieedd
diamine intermediate [17]. The addition of H4PteGlu to as substrate and from 0.8 ⋅ 104 to about 4.4 ⋅ 104s−1⋅M−1
glycine-saturatedwildtypeSHMTresultsintheincreaseof
the498nmabsorbingband,correspondingtothequinonoid when glycine was used as substrate; 𝑘on values were very
similar for both T254 mutant forms. After correction for
intermediate.Thelargeincreaseinabsorbancebelow400nm
the concentration of anionic substrate, 𝑘󸀠 values showed a
is the absorbance of the excess H4PteGlu. Instead, when on
10-folddecreaseaspHincreasedfrom6.4to8.0andranged
the T254A mutant was saturated with glycine, even in the
bpraensde.nThceeoefffHe4cPtsteoGftlhue,iatdddiditinoontosfhaoswataunryat4in9g8cnomncaebnstorrabtiionng 1fr3om⋅ 12006 ⋅to1016.8to⋅11.086⋅s−110⋅6Ms−−11⋅Mwi−t1hwgiltyhciLn-es.eThrinee saingdmofriodmal
of L-serine to T254A rcSHMT mutant enzyme are similar dependence of 𝑘o󸀠n on pH suggests the titration of a group
tothoseobservedforglycine,withadecreaseofthe430nm at the active site involved in a general acid catalysis, with
absorbing band and a concomitant increase of the 343nm a pK𝑎 around 6.8 (Figure2). If the internal aldimine of the
absorbing species. In contrast, when wild type rcSHMT is mutant enzymes is mostly in the carbinolamine form, as
saturated with L-serine, there are a marked increase and inferred from the absorption spectra shown in Figure1, it
a blue shift of the major absorbing band, centered now at mustbedehydrated(throughtheprotonationofthehydroxyl
426nm,butnoabsorbanceisobservedataround340. group followed by the elimination of water) in order to be
ThespectralfeaturesoftheunligandedT224Cmutantare convertedintotheketoenaminethatisabletoreactwiththe
largely similar to those of the wild type enzyme. However, incoming amino group of the substrates. Importantly, we
the 340nm band is slightly more intense. If compared to haveobservedthattheratioofthe428nmand340nmbands
theT254Amutant,therelativeintensitiesofthe430nmand changeswithpHinasimilarmanner,withtheketoenamine
340nmbandsarereversed.Spectralchangesuponsubstrates formbeingfavoredathigherpHvalues(datanotshown).A
addition are similar to the ones observed with the T254A possible candidate for this acid catalysis is Tyr73, which is
mutant, except that a residual absorbance is shown in the involved in a cation-𝜋 interaction with Arg263. It has been
420–430nmregion. shown that Tyr residues involved in cation-𝜋 interactions
mayhavetheirpK𝑎 loweredby1to3pHunits(thenormal
2.2.KineticsStudies. ThespectralpropertiesofthercSHMT pK𝑎 of tyrosine residue is about 9.5) [19]. Furthermore,
T254A and T254C mutant enzymes described above sug- this residue points toward the protein region where the
gest that the gem-diamine intermediate accumulates upon transaldimination reaction takes place, and its phenolic
󸀠
substrateaddition.Thepurifiedmutantenzymesweretested hydroxylislocatedincloseproximitytoC4 (seeFigure6).
for catalytic activity using L-serine and L-allo-threonine When L-serine was used as substrate, 𝑘off values showed a
as substrates (Table1). For L-serine, both mutant enzymes decreasingtrendaspHwasincreasedandrangedfrom10.3
showed slightly increased 𝐾𝑚 values (about 2-fold) when to3.1s−1fortheT254Amutantformandfrom39to23s−1for
comparedtowildtypercSHMT;𝑘catvaluesdecreased46-fold T254C.Incontrast,whenglycinewasusedassubstrate,𝑘off
4 BioMedResearchInternational
0.16 0.16
No ligand Wild type
Wild type
0.12 +gly 0.12 +L-ser
+H4PteGlu
nce nce
bsorba 0.08 +gly bsorba 0.08 No ligand
A A
0.04 0.04
0.00 0.00
300 350 400 450 500 300 350 400 450 500 550
Wavelength (nm) Wavelength (nm)
(a) (b)
+gly T254A T254A
0.08 0.08 +L-ser
nce 0.06 +H+g4lPyteGlu nce 0.06
ba ba
bsor 0.04 bsor 0.04
A A
No ligand
0.02 No ligand 0.02
0.00 0.00
300 350 400 450 500 300 350 400 450 500 550
Wavelength (nm) Wavelength (nm)
(c) (d)
T254C T254C
+gly +gly
0.08 +H4PteGlu 0.08
+L-ser
0.06 0.06
nce No ligand nce
ba ba No ligand
or 0.04 or 0.04
bs bs
A A
0.02 0.02
0.00 0.00
300 350 400 450 500 300 350 400 450 500 550
Wavelength (nm) Wavelength (nm)
(e) (f)
Figure1:AbsorptionspectraofwildtypeandmutantrcSHMTintheabsenceandpresenceofsubstrates.Leftpanelsshowtheabsorption
∘
spectraofwildtype,T254AandT254CrcSHMTsat30Cbefore(blacklines)andaftertheadditionof90%saturatingglycine(bluelines).
Greenlinesarespectratakenaftertheadditionof100𝜇MH4PteGlutothesamplescontainingglycine.Rightpanelsshowtheabsorption
spectraofthesameenzymesbefore(blacklines)andaftertheadditionof90%saturatingL-serine(redlines).
values slightly increased with pH, ranging from 1 to 2.9s−1 the𝑘offvaluesdeterminedwiththemutantenzymesareabout
fortheT254Amutantformandfrom5.1to7.0s−1forT254C. 2ordersofmagnitudelowerthanthewildtype. Thesedata
Stopped-flowstudiesdonemanyyearsagoatpH7.3for suggestthatthemutationshavenotsignificantlyaffectedthe
wild type rcSHMT [12] gave a comparable 𝑘on value to the rateofformationofthegem-diamineintermediatebuthavea
valuesfortheT254AandT254Cmutants.Ontheotherhand, moresignificanteffectontherateofitsbreakdown.
BioMedResearchInternational 5
25
T254A T254C
20 20
)
610 60)
1× 15 ×1 15
−M −1
M
1
−(sn10 −1(s10
󳰀ko 󳰀kon
5 5
0 0
6.0 6.5 7.0 7.5 8.0 8.5 9.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH pH
(a) (b)
Figure2:Dependenceof𝑘󸀠 valuesdeterminedfromstopped-flowexperimentsonpH,forT254AandT254CrcSHMTmutants.Opencircles
on
correspondtodataobtainedwithL-serine,whileclosedcirclescorrespondtodataobtainedwithglycine.Thecontinuouslinesthroughthe
experimentalpointswereobtainedthroughaleastsquareminimizationprocessusingtheequationforasigmoidalcurve(thesoftwareused
wasGraphPad,Prism).
Table 1: Kinetic constants for the hydroxymethyltransferase and external aldimine. These two hypotheses are not mutually
retro-aldol cleavage reactions catalyzed by wild type and T254 exclusiveandcouldbeappliedatthesametime.
mutantrcSHMTforms.
Kinetic FormofrabbitcytosolicSHMT 2.3.CrystalStructures. Wedeterminedthecrystalstructures
Substrate
constant Wildtype T253A T253C oftheT254AandT254Cmutantenzymesandthecomplex
L-Serine 𝐾𝑚(mM) 0.3 0.6 0.5 ofeachwithglycineandL-serine,andthestructureofwild
𝑘 (min−1) 850 18.5 1.6 type rcSHMT to a higher resolution than had previously
cat been attained [20]. Table3 lists the crystallographic and
L-allo-Threonine 𝐾𝑚(mM) 1.5 2.5 2.6 refinementdataforthenewrabbitcytosolicSHMTstructures
𝑘cat(min−1) 130 14.3 4.5 determinedanddescribedinthiswork.Allstructuresexcept
for the T254C-glycine complex were solved in space group
P41withatetramerof4independentlydeterminedsubunits
perasymmetricunit.TheT254C-glycinecomplexwassolved
If the rapid spectroscopic changes occurring when the
enzyme was mixed with saturating concentration of sub- inspacegroupP41212withadimerperasymmetricunit.The
oligomericstructureofrcSHMTconsistsoftwotightdimers
strates were followed for a longer period of time (up to 50
each made of identical monomers. These tight dimers are
seconds),aslowfirst-orderincreasewasobservedat426nm.
looselyassociatedtoformaso-calleddimerofdimers.Disor-
For the T254A mutant with saturating concentration of L-
−1 dereddensityrecurredintheinsertsegmentaroundresidue
serine,therateconstantwasdeterminedtobe10min .This
272 and at the amino termini of all monomer subunits, as
value is in agreement with previous studies on the T226A
observed in previously determined SHMT structures, but
mutant of ecSHMT [17]. It is interesting to notice that this
rate constant is very close to the 𝑘 measured for both thesedisorderedresiduesarenotclosetotheregionsofthe
cat enzymearoundthePLPandsubstratebindingsite,whichare
hydroxymethyltransferaseandretro-aldolcleavagereactions
thesubjectofthisstudy.
catalyzed by this mutant. This shows that for the T254A
mutanttheconversionofthegem-diamineintotheexternal
aldimineintermediatehasbecometherate-limitingstep. 2.3.1. Differences in Structure of Unliganded Wild Type and
Thespectralandkineticstudiesbothstronglysuggestthat T254Mutants. Exceptforthesmalldifferencesintheactive
removing the hydroxymethyl group of Thr254 by replacing site described herein, all structures are virtually identical
it with either an Ala or a Cys residue greatly slows the (rmsd <0.4A˚ in all cases). The interactions of the PLP
conversionofthegem-diamineintermediatestotheexternal cofactorwiththeproteininthewildtypeandT254mutants
aldimine. This may be the result of either blocking the arealmostidentical.ThedistanceoftheAsp228carboxylate
conversionofgem-diamineItogem-diamineIIorofslowing toN1ofthepyridineringisunchangedinthemutantsasare
conversionofgem-diamineIItotheexternalaldimine.There- the noncovalent bond constraints on the phosphoryl group
fore,theslowingofthecatalyticcyclecouldbeexplainedby of the PLP tail and the coplanarity of the His148 imidazole
either the mutant gem-diamine complexes being in a more with the PLP ring. The active sites of all subunits of the
stable form (in an energy well) compared to the wild type wildtypehavephosphateorMESbufferanionsboundwhere
enzyme or by an increased energy barrier to pass to the the carboxylate moiety of the amino acid substrate binds
6 BioMedResearchInternational
Table2:Kineticconstantsfortherateofformationandbreakdownofthegem-diaminecomplexformutantrcSHMTformswithglycineand
L-serinesubstrates.
T253A T253C
Substrate pH
𝑘on(s−1M−1⋅104) 𝑘o󸀠n(s−1M−1⋅106) 𝑘off(s−1) 𝑘on(s−1M−1⋅104) 𝑘o󸀠n(s−1M−1⋅106) 𝑘off(s−1)
6.4 3.3 20 10.3 2.9 18 39
6.8 5.8 14 8.3 4.0 10 36
L-Serine 7.2 8.7 8.6 5.8 6.8 6.7 32
7.6 9.5 3.7 5.3 8.7 3.4 27
8.0 10.7 1.8 3.1 9.4 1.5 23
6.4 0.8 13 1.0 0.5 8.5 5.1
6.8 1.3 8.0 1.3 0.7 4.5 6.7
Glycine 7.2 2.1 5.0 1.9 1.5 3.8 6.9
7.6 3.0 2.9 2.4 2.1 2.2 7.0
8.0 4.4 1.8 2.9 3.7 1.6 6.8
in SHMT-substrate complexes (see next paragraph). In the insolution,theamountofthecanonical428nmketoenamine
T254Amutant,thissiteisoccupiedbyphosphateionsinone bandishigherthanintheT254Amutant.
dimerandbyMESintheotherdimer.IntheT254Cmutant
thisanionicsiteisoccupiedbywaterinthreesubunitsandby 2.3.2. The Glycine and L-Serine Complexes of T254A and
anapparentphosphateinthefourthsubunit(Table4). T254C Mutants. The structures of the T254A and T254C
There are significant differences between the wild type mutants with glycine and L-serine substrate ligands are all
and the T254 mutants in the conformation of the PLP inthegem-diamineform(Table4),showingboththeamino
methylenephosphatetailalthoughtheseareseentoadiffer- group of the substrate and the 𝜀-amino group of Lys257
󸀠
entextentindifferentsubunits(Figure3).Thesedifferences formingbondstoC4 ofPLP.Inallsubunitsofbothmutants
correlate with the absence of the C𝛽 methyl group in the andwithbothsubstrates,theorientationofthePLPringand
Ala254 and Cys254 side chains in the mutants. In the wild the conformation of the methylene phosphate tail are very
typestructure,thismethylgroupofThr254projectstoward similar.Interestingly,theinhomogeneityseenintheinternal
themethylenephosphatetail.Replacementofthesidechain aldiminestructuresofthemutantenzymeshasdisappeared
by Ala or Cys opens a small space that allows the cofactor upon binding of substrates. For example, Figure4 shows a
󸀠
tail to assume a different conformation, moving the C5 comparisonbetweentheactivesitestructuresoftheT254A
󸀠
towardsthiscavity.Thisisthemostevidentchangeobserved mutantwitheitherL-serine(a)orglycine(b)boundtoC4
in the mutants. The fact that not all of the T254A and C ofPLPinthegem-diamineformandtheinternalaldimine.
subunits show this change to the same extent suggests that Thecomparisonismadewiththesubunitoftheunliganded
there is a low barrier to this switch of conformations and mutant enzyme in which the internal aldimine shows the
thatthecrystallizationprocessisselectingoutanasymmetric largest difference with respect to wild type (Figure3(d)).
distribution. As a consequence of the elbow-like rotation It can be seen that, in both gem-diamine structures, the
of the methylene phosphate PLP tail in the mutant T254 orientation of the PLP ring and the conformation of the
forms,thePLPringorientationalsodivergesfromthatofthe methylenephosphatetailandoftheactivesitelysine(Lys257)
wild type. Moreover, in the mutant enzyme structures with are very similar to those of the internal aldimine form and
thelargestPLPtailconformationaldifferences,thealdimine therefore differ significantly from the wild type internal
linkagebetweenC4󸀠andthe𝜀-aminogroupoftheactivesite aldimine structure. The carboxylate group of substrates is
lysine(Lys257)isfarfrombeingintheplaneofthePLPring oriented to form dual hydrogen bonds with Arg402 and
(Figure3(d)), as required by the double bond conjugation also with Y83. Residues Ser203 and His231 make hydrogen
󸀠
present in the ketoenamine form of the internal aldimine. bondswiththeO3 ofPLP, asalreadyobserved inallother
As the aldimine linkage is driven away from this plane, its SHMTstructures.IntheL-serinegem-diaminecomplex,the
conjugation with the 𝜋-electron system of the pyrimidine hydroxylgroupofthesubstratemakesH-bondinteractions
ringisdiminished,andthemaximumabsorbanceisshiftedto withGlu75andTyr83,asalsoobservedinthecrystalstructure
lowerwavelengths[21],asobservedforthemutantenzymes of the Bacillus stearothermophilus SHMT-L-serine complex
insolution(Figure1).Asdiscussedabove,wecannotexclude [22].
that the 340nm absorbing band of the unliganded mutant Wild type rcSHMT enzyme cocrystallized with glycine
enzymesmaycorrespondtothehydratedformoftheinternal and5-CHO-H4PteGlushowedtwooftheenzymesubunitsin
aldimine(carbinolamine;Scheme1).Intheinternalaldimine thegem-diamineform[19](Table4).Noexternalaldimines
formoftheT254Cmutant,theconformationalchangeofthe werepresent,astheothertwosubunitswereintheinternal
methylene phosphate tail and the displacement of the PLP aldimine form. It is worth noting that structural variation
ringarelessevident(datanotshown).Thiscorrelateswiththe amongsubunitsinligandbindingsiteoccupancyandtypeof
observationthat,intheabsorptionspectrumofthismutant intermediatecomplexisalsofoundinothereukaryoticand
BioMedResearchInternational 7
)
5
5
T254C+L-ser.114.0,114.0,155.6P411.8–2.55(2.62–2.90.9(76.9).202(.284).263(.362)52787(5963)5963(375)3840.1 0.0071.16 142712812102.00(96%)
1
1
)
5
6
hiswork. T254C+gly.114.6,114.6,156.922P41190.0–2.65(2.72–2.69.6(62.0).221(.333).305(.414)19976(1244)1654(96)36.6NA 0.0061.08 70269672.24(93%)
t
in 0)
d 4
ucturesdetermine T254C114.0,114.0,154.7P4111.8–2.40(2.46–2.90.1(86.9).195(.242).256(.346)62364(4393)7009(470)39.441.9 0.0061.13 14351331551.81(97%)
r 1
st
T 0)
statisticsforrcSHM T254A+L-ser.115.5,115.5,156.3P41115–2.40(2.46–2.488.3(85.8).197(.247).258(.318)63363(4478)7156(510)37.337.3 0.0061.13 14251461251.88(96%)
nt )
ctionandrefineme T254A+gly.115.0,115.0,156.8P410.0–2.65(2.72–2.6599.6(99.8).203(.292).277(.399)52905(3769)5968(406)3434.6 0.0071.22 1403142992.04(97%)
e 2
oll
c 5)
a 5
allographicdat T254A115.1,115.1,157.4P418–2.55(2.62–2.92.2(87.9).201(.306).271(.432)55295(3849)6204(426)44.843.8 0.0071.24 1424142872.04(96%)
st 1.
y 1
r 1
C
3: 0)
Table Wildtype5.6,115.6,156.0P41–2.10(2.16–2.198.5(98.2).199(.248).242(.316)05631(7668)11736(845)30.839.8 0.0081.3 14601231331.73(93%)
1 0 1
1 9.
1
1
d
ed
Cell/spacegroup ResolutionCompleteness(%)𝑅work𝑅free𝑁work𝑁free⟩⟨𝐵protein⟩⟨𝐵waterRMSDfromidealBondlengthsBondanglesRamachandranplotFavoredAdditionallyallowGenerouslyalloweForbiddenMolProbityscore
8 BioMedResearchInternational
Thr254/Ala254
Thr254/Ala254
Lys257
Lys257
(a) (b)
Thr254/Ala254 Thr254/Ala254
Lys257
Lys257
(c) (d)
Figure3:SuperpositionoftheactivesitestructuresofrcSHMTwildtype(salmon)andT254A(cyan)intheinternalaldimineform.Panels
(a)to(d)correspondtodifferentsubunitsofthetetramer.
prokaryoticSHMTs[22–26].TheabovewildtypercSHMT- exchange required to interconvert the two gem-diamine
glycine-5-CHO-H4PteGlu ternary complex structure and intermediates(seebelowfordiscussion).
thatofmousecytosolicSHMT(whichwasalsococrystallized Inthewildtypegem-diamine,thebondbetweenthesub-
󸀠
with glycine and 5-CHO-H4PteGlu [25]) are the only wild strateaminogroupandC4 ofPLPliesroughlyperpendicular
typeSHMTstructuresinwhichgem-diamineintermediates to the cofactor ring, indicating a strong similarity to gem-
canbeseen. diamine I intermediate, in which the amino group of the
󸀠
When wild type and T254A rcSHMT-glycine gem- substrate has attacked the C4 Schiff base of the cofactor
diamine structures are superimposed, some striking differ- with a trajectory that is perpendicular to the pyridine ring
ences can be noticed (Figure5). The methylene phosphate (Scheme1). It appears that the wild type rcSHMT in the
tail conformation and the orientation of the PLP ring are crystal has been trapped as a gem-diamine I intermediate
significantly different. The carboxylate group of glycine in that barely accumulates in solution. On the other hand,
wild type rcSHMT gem-diamine, although still interacting the gem-diamine forms found in T254 mutants seem to
with Y83 and Arg402 through one of its oxygen atoms, is be in an aberrant position. The best way to appreciate this
nomoreorientedtointeractoptimallywithArg402,andthe is to superimpose the structures of wild type gem-diamine
other oxygen points away from it. Moreover, the position (Figure6(a))orT254mutantgem-diamine(Figure6(b))with
of the two amino groups in the gem-diamine is different wild type internal and external aldimines. In SHMTs, as
between wild type and mutant (see arrows in Figure5). In PLP reacts with the substrate and the internal aldimine is
particular, the hydrogen atoms of the amino groups point convertedintotheexternalaldimine,thepyridineringrotates
∘
away from Tyr73, which might be involved in the proton by about 25 , primarily around the C2-C5 axis. This is also
BioMedResearchInternational 9
Tyr83
Tyr83
Glu75
Glu75
Arg402 Arg402
Lys257
Lys257
Ser203
Ser203
His231
His231
Ala254 Ala254
(a) (b)
Figure4:SuperpositionoftheactivesitestructuresoftheT254Amutantintheinternalaldimineform(cyan)andasagem-diaminecomplex
(orange)witheitherL-serine(a)orglycine(b).
of the PLP ring is also observed in the unliganded forms
ofT254AandT254CrcSHMTstructures.Thegem-diamine
structures of the mutant enzyme appear to be close to the
gem-diamine II intermediate (Scheme1), and the angle of
Tyr83 thebondfromC4󸀠 ofthePLPringtothe𝜀-aminogroupof
Tyr73 ∘
K257 being close to the 90 predicted for this intermediate
II,inwhichtheaminogroupofLys257hastobeeliminated
in order to form the external aldimine intermediate. More-
over, the carboxylate group of the substrate makes optimal
dual hydrogen bonds with Arg402 (Figure6(b); structure
in orange), as is observed in the external aldimine form of
Arg402 wild type SHMT-glycine complex (Figure6(b); structure in
magenta).
These observations suggest that in the T254 rcSHMT
crystals, the PLP-substrate complex is blocked in the form
of an anomalous and stable gem-diamine intermediate. As
Thr254/Ala254
mentioned above, it is noteworthy that in the T254 mutant
gem-diamine intermediates the position and orientation of
Figure5:Superpositionoftheactivesitestructuresofthewildtype the two amino groups point away from Tyr73 compared to
(slate)andT254Amutantform(orange)ofrcSHMTasgem-diamine thewildtypegem-diaminestructure.Tyrosine73wasshown
complexes with glycine (pdb 1ls3). The arrows in the figure point in E. coli and bsSHMT to have an important role in the
towardstheaminogroupsofthegem-diaminecomplexes. transaldiminationprocessandwasproposedtoactasproton
exchangerbetweengem-diaminesIandII.Interestingly,Tyr
to Phe mutants of this residue also accumulate the gem-
observed in prokaryotic B. stearothermophilus SHMT with diamineintermediate[18,27].
both glycine and L-serine as substrates and in the ternary An additional remarkable variation in the T254 mutant
complexwithglycineand5-CHO-H4PteGlu[22].Inthewild structurescomparedtowildtypeisobservedinthepeptide
type rcSHMT gem-diamine, the PLP ring lies between the bond between His256 and Lys257. Among the determined
positions observed in the internal and external aldimines. mutant structures, this peptide bond adopts two distinct
Strikingly,inthemutantT254gem-diaminethecofactorring orientations, one similar to the wild type rcSHMT and
∘
has a different orientation, which is unlikely to correspond the other differing by up to 180 . The local variation in
to that occurring in the transaldimination reaction. This is conformation of the His256-Lys257 peptide bond may be
obviouslyaconsequenceofthemutation,sincethisposition linkedtothercSHMTT254mutations,since,intheinternal
10 BioMedResearchInternational
Tyr83
Tyr73 Tyr83 Tyr73
Lys257 Lys257
Arg402 Arg402
(a) (b)
Figure6:Superpositionofwildtypestructurescorrespondingtotheinternalaldimine(salmon;rcSHMT)andexternalaldimine(magenta;
mcSHMT,pdb1eji)formsofthecofactorwiththeglycinegem-diamineformofthewildtype(slate)andT254Amutant(orange).
aldimine of wild type rcSHMT, the carbonyl oxygen of variesinthemutants.TheselocalstructurechangesinT254
His256, which is flipped in some mutant structures, makes mutantscouldberesponsibleforaperturbationoftheproton
a hydrogen bond through a water molecule to the Thr254 transferchainwhichslowsthetransaldiminationreaction.
side chain. The lack of uniformity in structure among the Available external aldimine SHMT structures (such as,
subunits of the T254 mutants in the conformation of the E.coliSHMTasternarycomplexwithglycineand5-CHO-
PLPmethylenephosphatetail,thepositionofthePLPring, H4PteGlu(pdb1dfo)andbsSHMTwithglycineandL-serine
and the orientation of the His256-Lys257 peptide bond (pdb 1kl1 and 1kkp, resp.)) all show a good hydrogen bond
suggest that mutation of T254 relaxes some local structural betweentheLys257𝜀-aminogroupandthehydroxylgroupof
constraintsaroundtheactivesite. theThr254sidechain,asoriginallysuggestedbyPascarellaet
al.[16].Thenucleophilicityofthe𝜀-aminogroupandtheNH2
ofthesubstratemustbebalancedsothattransaldimination
3.Conclusions
canoccurrapidlyfromeitherdirection.Thehydrogenbond
between Thr254 and the Lys257 𝜀-amino group is likely to
The T254A and T254C mutations have created a small
be a critical determinant of this balance. In the T254A and
empty space in the active site of rcSHMT due to the
T254Cmutants,whichlackthehydroxylgroup,thisH-bond
absence of the threonine methyl group. This has allowed
cannotbeformed.Wesuggestthatthelossofthishydrogen
the methylene phosphate tail of PLP to adopt a stable,
bonddestabilizestheexternalaldimineoftheT254mutants
uncharacteristicconformationthatisinturnresponsiblefor
relativetothegem-diamineintermediatesandresultsinthe
an aberrant positioning of the PLP ring. This clearly affects
trappingofthelatter.
thetransaldiminationstepoftheSHMTreaction,makingit
PLP-dependentenzymestypicallycatalyzethereversible
therate-limitingstepinthecatalyticcycle.Inthesemutants,
transaldiminationreactionbetweentheinternalandexternal
wheneitherL-serineorglycineisaddedtotheenzyme,gem-
aldiminesveryrapidly,withinthedeadtimeofstopped-flow
diamine intermediates greatly accumulate. This may be the
measurements, and do not accumulate gem-diamine inter-
resultofeitherblockingtheconversionofgem-diamineIto
mediates.Thisisacrucialstepinthecatalyticmechanismof
gem-diamineII,slowingtheconversionofgem-diamineIIto
allPLP-dependentenzymesbecauseitdeterminesbindingof
theexternalaldimine,orboth.
substrates and release of products. Our studies show that a
Conversion of the gem-diamine II into the external
aldiminerequiresthatthe𝜀-aminogroupofLys257isproto- single,semiconservativemutationofanactivesiteresiduecan
󸀠 becriticalforthetransaldiminationinSHMT.
natedandthatitleavesperpendicularlyfromthesifaceofC4
ofPLP.InScheme1thisisshownasadirectprotontransfer
from the substrate amino group in gem-diamine I to the 4.MaterialsandMethods
leavingaminogroupofLys257ingem-diamineII.However,
thisprotontransferalmostcertainlydoesnotoccurdirectly 4.1. Materials. All chemicals, coenzymes, antibiotics, and
butthroughshiftsofprotonsinanetworkofacid-basegroups buffers were from Sigma-Aldrich (St. Louis, MO, USA)
at the active site. It is possible, based on the wild type and or FisherScientific (Pittsburgh, PA, USA). (6S)-H4PteGlu
T254mutantstructures,thatprotonationoftheLys257amine and (6S)-5-CHO-H4PteGlu were gifts from Merck Eprova
ofthegem-diamineismediatedbyTyr73.Thepositionofthe AG(Schaffhausen,Switzerland).Crystallizationbufferswere
gem-diamine amino groups relative to the Tyr73 side chain fromHamptonResearch(LagunaNiguel,CA,USA).