Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus

15 Pages·2017·5.09 MB·English

molecules
Article
Functional Impact of the N-terminal Arm of Proline
Dehydrogenase from Thermus thermophilus
MiekeM.E.Huijbers1,IlonavanAlen1,JennyW.Wu1,ArjanBarendregt2,3,
AlbertJ.R.Heck2,3 ID andWillemJ.H.vanBerkel1,* ID
1 LaboratoryofBiochemistry,WageningenUniversity&Research,Stippeneng4,6708WEWageningen,
TheNetherlands;[email protected](M.M.E.H.);[email protected](I.v.A.);
[email protected](J.W.W.)
2 BiomolecularMassSpectrometryandProteomics,BijvoetCenterforBiomolecularResearchandUtrecht
InstituteofPharmaceuticalSciences,UtrechtUniversity,Padualaan8,3584Utrecht,TheNetherlands;
[email protected](A.B.);[email protected](A.J.R.H.)
3 NetherlandsProteomicsCenter,Padualaan8,3584Utrecht,TheNetherlands
* Correspondence:[email protected];Tel.:[phone]
Received:8December2017;Accepted:14January2018;Published:16January2018
Abstract:Prolinedehydrogenase(ProDH)isaubiquitousflavoenzymethatcatalyzestheoxidationof
prolineto∆1-pyrroline-5-carboxylate. ThermusthermophilusProDH(TtProDH)containsinadditionto
itsflavin-bindingdomainanN-terminalarm,consistingofhelicesαA,αB,andαC.Here,wereport
the biochemical properties of the helical arm truncated TtProDH variants ∆A, ∆AB, and ∆ABC,
producedwithmaltose-bindingproteinassolubilitytag. Allthreetruncatedvariantsshowsimilar
spectralpropertiesasTtProDH,indicativeofaconservedflavin-bindingpocket. ∆Aand∆ABare
highlyactivetetramersthatrapidlyreactwiththesuicideinhibitorN-propargylglycine. Removal
oftheentireN-terminalarm(∆ABC)resultsinbarelyactivedimersthatareincapableofforminga
flavinadductwithN-propargylglycine. CharacterizationofV32D,Y35F,andV36Dvariantsof∆AB
establishedthatahydrophobicpatchbetweenhelixαCandhelixα8iscriticalforTtProDHcatalysis
andtetramerstabilization.
Keywords: flavoprotein; proline dehydrogenase; protein engineering; protein oligomerization;
solubilitytag;suicideinhibition;TIM-barrel
1. Introduction
Prolinedehydrogenase(ProDH;EC1.5.5.2)isaubiquitousenzymeinvolvedinprolinecatabolism.
ProDH catalyzes the flavin-dependent oxidation of L-proline to ∆1-pyrroline-5-carboxylate (P5C).
AfterP5Chydrolysis,theresultingglutamicsemialdehyde(GSA)isoxidizedtoglutamatethroughthe
actionof∆1-pyrroline-5-carboxylatedehydrogenase(P5CDH;EC1.2.1.88)(Scheme1). ProDHand
P5CDHexistasseparatemonofunctionalenzymesineukaryotesandsomebacteria,butarefusedinto
abifunctional[1,2]ortrifunctional[3]enzymeinotherbacteria. Inthesemulti-functionalenzymes,
called proline utilization A (PutA), the C-terminus of ProDH is fused to P5CDH, allowing for the
channelingoftheP5C/GSAintermediatebetweentheenzymes[4–9].
ProDHhasadistorted(βα) TIM-barrelfold(Figure1A),whichisconservedthroughoutthe
8
PutA/ProDHfamily[11,12]. OpposedtotheclassicTIM-barrelfold,theProDHbarrelbeginswith
a helix (α0) rather than a strand (Figure 1). This extra helix occupies the location that is normally
reservedforα8. Asaconsequence,α8isnotlocatedalongsideβ8,butontopofthebarrel[1,13,14].
Thedistortedlocationofα8iscrucialforcatalysis,sinceitcontributesthreestrictlyconservedresidues
(Tyr-x-x-Arg-Arg)thatinteractwiththesubstrate[13,14].
Molecules2018,23,184;doi:10.3390/molecules23010184 www.mdpi.com/journal/molecules
Molecules2018,23,184 2of15
Molecules 2018, 23, 184 2 of 15
Molecules 2018, 23, 184 2 of 15

SScchheemmee 11.. CoCnovnevresriosino nof oLf-pLr-oplrionlein teo tLo-gLlu-gtalumtaamte abtey bpyroplirnoel indeehdyedhroygdernogaseen a(sPero(DPrHo)D aHn)d aΔn1d-
p∆y1-rproylrirnoel-i5n-ec-a5r-bcaorxbyolaxtyel adteehdyedhryodgreongaesnea (sPe5(CPD5CHD).H S)c.hSecmheem baesbeads eodn o[1n0[]1. 0].
The NS-tcehremmien a1.l Cseonqvueersniocne ooff LP-proroDlinHe itso pL-ogolurtlaym caoten bsye rpvreodli.n eM doenhoydfurongcetniaosne a(lP reouDkHa)r yaondti cΔ 1P-roDHs,
TheNp-tyerrrmoliinnea-5l-csaerqbouxeynlactee doefhyPdrrooDgeHnasies (pPo5CoDrlHy).c Socnhesmerev beadse.dM ono [n10o]f. unctionaleukaryoticProDHs,
including the human enzyme [15], have an elongated N-terminus when compared to monofunctional
includingthehumanenzyme[15],haveanelongatedN-terminuswhencomparedtomonofunctional
bacterial PrTohDeH Ns- t[e1r1m,1in2a]l. sPerqouDenHc ef roofm Pr ToDheHrm isu sp othoerlrym coopnhsielurvs e(dT. tMProonDoHfu)n cctoionntaali nesu kaanr yNo-titce rPmroiDnaHl sa, rm
bacterialProDHs[11,12]. ProDHfromThermusthermophilus(TtProDH)containsanN-terminalarm
consistiinncglu odfi nthgr tehee hheulmicaens :e αnzAy,m αeB [,1 5a]n, dha αvCe a (nF eiglounrgea 1te)d. HNe-tleirxm αin8 ufsit ws hinetno c tohmep calreefdt, two mhiocnho ifsu nfocrtimoneadl by
consistingofthreehelices: αA,αB,andαC(Figure1). Helixα8fitsintothecleft,whichisformedby
helices bαacAte,r iαalB P raonDdH αs C[1.1 ,T12h]i. sP rcolDefHt ifsr oams sTuhmermedu s ttoh ebrme opinhivluosl v(TetdP rionD Hch) acnonnetaliinnsg a nP 5NC-t/eGrmSAin abl eatrwme en
helicesαA,αBandαC.ThiscleftisassumedtobeinvolvedinchannelingP5C/GSAbetweenTtProDH
TtProDcHon asinsdtin Tgt Pof5 tChDreHe h [e1l,i1ce6s]:. αA, αB, and αC (Figure 1). Helix α8 fits into the cleft, which is formed by
andTPtrPhe5evlCiiocDeusHs lαy[A1, ,,w1 α6e]B .s ahnodw αedC . thTahti sT ctlPefrto Dis Has sisu moveder ptor obdeu icnevdo livne dE sinch ecrhiacnhniae lcinolgi (PE5.C c/GolSi)A w bheetwn eietns N-
PrTetvPiroouDsHly ,anwde TtsPh5oCwDeHd [1t,h16a]t. TtProDH is overproduced in Escherichia coli (E. coli) when its
terminus is fused to maltose-binding protein (MBP) [17] and that the recombinant enzyme does not
N-terminusPirsefvuiosuedslyt,o wmea slthooswe-ebdi nthdaitn gTtpPrrooDteHin i(sM oBvePr)p[r1o7d]uacnedd itnh aEtstchheerircehciao mcoblii n(Ea.n ctoelin) zwyhmene ditso eNs-not
discriminate between FAD and FMN as cofactor [18]. Replacing Phe10 and Leu12 in helix αA with
discrimtienrmatienbues tiws feuesnedF AtoD maanltdosFeM-bNindaisngco pfraoctteoirn [(1M8B].PR) e[1p7l]a cainndg tPhahte t1h0e arnecdomLebuin1a2nitn ehnezylimxeα Adowesi tnhotG lu
Glu residues (further referred to as MBP-TtProDH variant EE) successfully released MBP-TtProDH
discriminate between FAD and FMN as cofactor [18]. Replacing Phe10 and Leu12 in helix αA with
residues(furtherreferredtoasMBP-TtProDHvariantEE)successfullyreleasedMBP-TtProDHfrom
from non-native self-association, yielding homogeneous tetramers [19]. Although TtProDH
Glu residues (further referred to as MBP-TtProDH variant EE) successfully released MBP-TtProDH
non-nativeself-association,yieldinghomogeneoustetramers[19]. AlthoughTtProDHcrystallizesasa
crystallfirzoems anso an -ndaimtiveer (sFelifg-ausrseo c1ia) ti[o1n],, ity iiesl dwinogr thhyom oof gnenoeteo utsh atte tirna msoerlus ti[o1n9],. thAel thenouzgyhm eT trPermoDaiHn s a
dimer(Figure1)[1],itisworthyofnotethatinsolution,theenzymeremainsatetramerafterremoval
tetramecrry asfttaellri zreesm aos vaa ld oimf tehr e( FMigBuPre s1o)l u[1b]i, liitt yi st awgo [r1th3y]. of note that in solution, the enzyme remains a
oftheMBPsolubilitytag[13].
tetramer after removal of the MBP solubility tag [13].

Figure 1. Structural features of Thermus thermophilus proline dehydrogenase (TtProDH). (A) Three-
Figure 1. Structural features of Thermus thermophilus proline dehydrogenase (TtProD H).
dimensional model of the crystal structure of the TtProDH dimer (PDB entry 2G37). The N-terminal
(A) Three-dimensional model of the crystal structure of the TtProDH dimer (PDB entry 2G37).
Figure h1e. liSctersu αctAu r(aglr efeena)t,u αreBs ( roefd T), haenrmd uαsC t h(berlumeo) pahriel uins dpircoatleinde, ads ewhyeldl raosg heenliaxs eα 0(T (tpPurropDleH) a)n. d(A t)h eT hCr-ee-
TdhimeeNn-sttieeorrnmmaiilnn amal lohhdeeleilxli coαef8s t(αhbeAr ocw(rgynrs)e;t ea(nBl ))s, tArαumBcit(nuroer dea c)o,idfa tnshedqe uαTeCtnPc(rebo loDufe HT)t aPdrrieomDienHrd .(i PcSaeDcteoBdn ed,naatrsryyw s t2erGlulc3at7su)rh.a eTl lheilxee mαN0e-nt(etpsr umornipn lael)
and the C-terminal helix α8 (brown); (B) Amino acid sequence of TtProDH. Secondary structural
helices tαopA o f( gthree esneq),u αenBc e( rheadv)e, tahne dsa αmCe c(obllourse )a sa irne Fiingduircea 1tAed. ,F iagsu rwe e1lAl iass b haseeldix o αn 0[1 (9p].u rple) and the C-
elementsontopofthesequencehavethesamecolorsasinFigure1A.Figure1Aisbasedon[19].
terminal helix α8 (brown); (B) Amino acid sequence of TtProDH. Secondary structural elements on
top of the sequence have the same colors as in Figure 1A. Figure 1A is based on [19].
Molecules 2018, 23, 184 3 of 15
To investigate the functional impact of the N-terminal arm of TtProDH in catalysis and
oligomerization in closer detail, we constructed MBP-fused variants, lacking, respectively, one (ΔA),
Mtwoloec u(ΔlesA2B01)8, ,o2r3 ,t1h8r4ee (ΔABC) N-terminal helices. ΔA and ΔAB turned out to be highly active tetram3oefr1s5,
while ΔABC was an almost inactive dimer. To further probe the role of helix αC, we addressed the
properties of site-specific variants V32D, Y35F, and V36D of ΔAB. This revealed that a hydrophobic
To investigate the functional impact of the N-terminal arm of TtProDH in catalysis and
oplaigtcohm beertiwzaeteinon hienlixcl αosCe randdet ahiell,iwx αe8c oisn sctrriuticctaeld foMr TBtPP-rfuoDseHd vcaatraialynstiss, alancdk itnegtr,armesepre scttaivbielliyz,aotinoen(. ∆A),
two(∆AB),orthree(∆ABC)N-terminalhelices. ∆Aand∆ABturnedouttobehighlyactivetetramers,
w2.h Rileesu∆lAtsB Cwasanalmostinactivedimer. TofurtherprobetheroleofhelixαC,weaddressedthe
propertiesofsite-specificvariantsV32D,Y35F,andV36Dof∆AB.Thisrevealedthatahydrophobic
2.1. Protein Expression and Purification
patchbetweenhelixαCandhelixα8iscriticalforTtProDHcatalysisandtetramerstabilization.
MBP-TtProDH EE, ΔA, ΔAB and ΔABC were overproduced in E. coli TOP10 cells. From 1 L of
2. Results
culture about 200–250 mg of each variant was purified, yields that we have described before for the
heterologous production of MBP-TtProDH [17,19]. From SDS-PAGE analysis of the purified
2.1. ProteinExpressionandPurification
enzymes, it can be appreciated that sequential removal of three helices results in a gradual decrease
of suMbuBnPit- TmtPorleocDuHlarE mE,a∆ssA (,F∆igAuBrea 2n)d. ∆ABCwereoverproducedinE.coliTOP10cells. From1Lof
cultuPrereavbioouutsl2y0, 0w–2e5 0dmemgoonfsetarachtevda rthiaantt rwemasopvuarl ifioef dt,hyei eMldsBPth afutwsioenh atvage dwesitchr ibtreydpbseinfo rdeofeosr tnhoet
hsiegtnerifoilcoagnotulys apfrfoecdtu tchteio cnatoaflyMtiBcP p-TrotPpreorDtieHs a[1n7d,1 o9l]i.gFormomeriSzDatSio-PnA bGeEhaavniaolry osifs TotfPtrhoeDpHu r[i1fi7e]d. Henowzyemveers,,
iatsc afonubneda wppitrhe cEiaEt e[d19t]h, attrysepqsuineonltyiasilsr eomf tohvea laormft-htrrueenchaetleicde svraersiaunlttss idnida gnroatd rueasludlte cinre ahsoemoofgseunbeuonuist
mproelpeacuralatiromnsa. sTsh(eFriegfuorree,2 w).e used the MBP-fused variants for further studies.

Figure 2. Purified MBP-TtProDH variants visualized on sodium dodecyl sulfate polyacrylamide gel
Figure2.PurifiedMBP-TtProDHvariantsvisualizedonsodiumdodecylsulfatepolyacrylamidegel
electrophoresis (SDS-PAGE). Molecular masses of marker proteins (M), from top to bottom: 100, 75,
electrophoresis(SDS-PAGE).Molecularmassesofmarkerproteins(M),fromtoptobottom:100,75,50,
50, 37, 25 kDa.
37,25kDa.
2.2. Spectral Properties
Previously, we demonstrated that removal of the MBP fusion tag with trypsin does not
The far-UV CD spectra of EE and the N-terminal variants are very similar (Figure 3A) and
significantlyaffectthecatalyticpropertiesandoligomerizationbehaviorofTtProDH[17]. However,
comparable to that of MBP-TtProDH [17]. The visible flavin absorption spectra of EE and the arm-
as found with EE [19], trypsinolysis of the arm-truncated variants did not result in homogeneous
truncated variants (Figure 3B) are also nearly identical to that of MBP-TtProDH [17,19], except that
preparations. Therefore,weusedtheMBP-fusedvariantsforfurtherstudies.
the lowest energy absorption band of ΔABC has shifted about 2 nm to higher wavelengths. These
2d.a2t.aS spuecptpraolrPt rtohpaetr ttihesere are no major structural changes and that deletion of the N-terminal helices
does not significantly alter the microenvironment of the flavin-binding site. A recent crystallographic
The far-UV CD spectra of EE and the N-terminal variants are very similar (Figure 3A) and
study confirmed that ΔABC (residues 38–279; PDB 5M42) has a similar overall structure as TtProDH
comparable to that of MBP-TtProDH [17]. The visible flavin absorption spectra of EE and the
(PDB 2G37) with an rmsd value of 0.338 Å (for 221 Cα atoms of A chains) [1,18].
arm-truncatedvariants(Figure3B)arealsonearlyidenticaltothatofMBP-TtProDH[17,19],except
thatthelowestenergyabsorptionbandof∆ABChasshiftedabout2nmtohigherwavelengths. These
datasupportthattherearenomajorstructuralchangesandthatdeletionoftheN-terminalhelices
doesnotsignificantlyalterthemicroenvironmentoftheflavin-bindingsite. Arecentcrystallographic
studyconfirmedthat∆ABC(residues38–279;PDB5M42)hasasimilaroverallstructureasTtProDH
(PDB2G37)withanrmsdvalueof0.338Å(for221CαatomsofAchains)[1,18].
MolecuMleosl2ec0u1le8s, 22031,81, 8243, 184 4 of 154 of15
Molecules 2018, 23, 184 4 of 15

Figure 3. Spectral properties of EE, ΔA, ΔAB, and ΔABC. (A) Far-UV CD spectra; (B) Visible flavin
absorption spectra.
F2Fi.ig3g.u uHrreyed3 3r..o SdSpypenecactmtrraiacll Ppprrrooopppeerertrtiteiiese ss ooff EEEE,, ∆ΔAA,, ∆ΔAABB,, aanndd ∆ΔAABBCC.. ((AA)) FFaarr--UUVV CCDD ssppeeccttrraa;; ((BB)) VVisisibiblleefl falavvinin
aabbssoorrppttioionns sppeeccttrraa..
EE purifies as a tetramer [17,19]. The truncated variants ΔA and ΔAB also form tetramers as
indicated by size exclusion chromatography (Figure 4A). ΔABC elutes mainly as a dimer (Figure 4A),
22.3.3.. HHyyddrrooddyynnaammiicc PPrrooppeerrttieiess
suggesting that αC plays a role in the tetramerization process. Previously, we showed that TtProDH
EpEErEep ppauurreridifif fieresosm aas sM aaBt Pete-tTrtrataPmmroeeDrrH [1[ 17a7l,s1,1o99 ]f.]o.r TmThhse ete tttrrruaunmncceaartste,e dbdu vvt atahrriaiaatn nthttses ∆aΔdAAdi atainondnd o ∆Δf A1A BMB a GallsusooH fCfoolr rimnmd tuteectetrrsaa mtmheee rrss aass
iinnddiiccafaotteremddab btyiyons si izozefe d eeixmxcclelurusss i[io1on7n]. c cThhhrroeo mcmrayatstotoagglr srataprpuhhcytyu( r(FeFi igaglusurore es4h 4AoAw)).s.∆ ΔaA AdBiBmCCer eeilcluu sttteresusc mmtuaarieinn (llcyyf. a aFssig aau ddreiim m1Aeer)r. ( (FFiigguurree 44AA)),,
Mixing of EE and ΔABC followed by analytical gel filtration reveals two separate peaks, a peak
ssuuggggeessttiinngg tthhaatt ααCC ppllaayyss aa rroollee iinn tthhee tteettrraammeerriizzaattiioonn pprroocceessss.. PPrreevviioouussllyy,,w wees shhoowweeddt thhaattT TttPPrrooDDHH
for the tetrameric species of EE and a peak for the dimeric species of ΔABC (Figure 4B). This indicates
pprreeppaarreeddf frroommM MBBPP--TTttPPrrooDDHHa alslsoof foorrmmsst teettrraammeerrss,,b buuttt thhaattt thheea addddiittiioonno off1 1M MG GuuHHCClli inndduucceess tthhee
that mixing of the two enzyme forms does not lead to EE-ΔABC association. Moreover, SDS-PAGE
ffoorrmmaattiioonno offd diimmeerrss[ [1177]].. TThhee ccrryyssttaall ssttrruuccttuurreea allssoos shhoowwssa ad dimimeerricics strtruucctuturree( c(cf.f.F Figiguurree1 1AA).).
of the gel filtration fractions shows that no subunit exchange occurs (inset Figure 4B).
Mixing of EE and ΔABC followed by analytical gel filtration reveals two separate peaks, a peak
for the tetrameric species of EE and a peak for the dimeric species of ΔABC (Figure 4B). This indicates
that mixing of the two enzyme forms does not lead to EE-ΔABC association. Moreover, SDS-PAGE
of the gel filtration fractions shows that no subunit exchange occurs (inset Figure 4B).

FigurFeig4u.reH 4y. Hdryoddroydnyanmamicicp prrooppeerrttiieess ooff EEEE, Δ,A∆,A Δ,A∆BA, aBn,da ΔnAdB∆C AasB mCoansitomreodn bityo SreudpebrdyexS u20p0e sridzee-x 200
size-eexxcclluussiioonn cchhrroommaattooggrarapphhy.y .(A(A) )EElultuiotino npapttaetrtne ronf otfheth MeBMPB-TPt-PTrtoPDrHoD vHarivaanrtisa; n(Bts); H(By)dHroyddyrnoadmyinc amic
propeprrtoiepserotfieEs Eofa EnEd a∆ndA ΔBACB,Cm, imxeixdedin ine qequuaalla ammoouunnttss aanndd eqequuiliiblirbarteadte adt raotormoo tmemtpeemraptuerrea touvreernoivgehrt.n ight.
SDS-PAGE of the gel filtration fractions is also shown.
SDS-PAGEofthegelfiltrationfractionsisalsoshown.

Additional information about the oligomeric state of the different variants was obtained by
MnFaiigtxiuvinree g 4mo. afHsEsy Edsraponedcdytrn∆oamAmeBictCr py.rf ooTpllheorewt ieeessd toimbf yEaEtae,nd Δ aAlmy, atΔiscAsaeBls, g a(eTnladfib ΔllteAr aB1t)Ci o canosn rmefivormenai tlosthrteawdt obΔysA eS puaapnredartd eeΔxpA e2Ba0 0ke ssx,iizaset -peak
forthpeerxtecedltuorsamimoinne arcnihctrlyosp maeasc tioteegstrraoapmfhEeyEr.s ,a( Anan)d dEa lΔuptAeioaBnkC p faoasrt tetdhrinme deorfi. m tWheeri tiMhc sBΔpPAe-TcBitCePs,r oomDfiHn∆o Arv BaarCmiao(nFutsing; tu(sB roe) fH 4tBeyt)dr.arTomhdeiysrnsi anamdreiic c ates
thatmpprirxeosipneengrtt,oi eifnst ohafge ErteEwe amoneden nΔt zAwyBimtChe, tmfhoeirx maendsa ilndy oetiqecusalan lgo aetml lfeoilautdrnatttsio oannE dEr ee-s∆quuAlitlsBi bC(rFaiagtseusdro eac t4i raAotio)o.m nF o.terM mΔopAre eraoantvuder rΔe, AoSvDBe,S rsn-oPigmAheGt . Eof
thegedSliDmfiSle-trPrsaA taGiroEen ooffbr atshecerti vgoeendls fwislthirtoahtw ionsnat tfhirvaaect tmniooanssss ui sbs apulesncoit trsoehmxocwehtnray.n , gaendo cfcourr sΔ(AinBsCe tmFoignuormee4rsB, ).although their
abundance is rather low. Denaturing the different complexes enabled an accurate mass measurement
Additionalinformationabouttheoligomericstateofthedifferentvariantswasobtainedbynative
mass Aspdedcittrioomnaelt riyn.fTohrmeeastitoimn aatbedoumt athssee so(liTgaobmlee1r)icc osntafitrem oft htahte∆ dAifafenrden∆t AvBareixainsttsp wreadso mobitnaainnetldy absy
tnetartaivmee rms,aasns ds∆pAecBtrComasetdriym. eTr.hWe iethsti∆mAaBteCd, mminasosreas m(oTuabnltes o1f) tectoranmfiremrs athreatp rΔesAe nat,nidn aΔgAreBe meexnistt
wpirtehdothmeinananaltylyti caasl tgeetrlafimlterrast,i oanndre sΔuAltBsC(F aigsu dreim4eAr). .WFoitrh∆ ΔAAaBnCd, ∆mAinBo,rs oammeoudnimts eorsf aterteraombseerrsv aedre
wpirtehsennatt,i vine amgaresesmspeenctt rwoimthe ttrhye, aanndalyfotirca∆l AgBelC fimltroantioomn erress,uallttsh (oFuigguhrteh 4eAir)a. bFuonr dΔaAn caenids ΔraAthBe, rsloomwe.
dimers are observed with native mass spectrometry, and for ΔABC monomers, although their
abundance is rather low. Denaturing the different complexes enabled an accurate mass measurement
Molecules2018,23,184 5of15
Denaturingthedifferentcomplexesenabledanaccuratemassmeasurementoftheindividualsubunits
(Table1). Thesemassesshowthatallofthevariantsexistofidenticalsubunits.
Table1.MolecularmassesofEE,∆A,∆AB,and∆ABCasdeterminedbynativeanddenaturedESI-MS.
Molecules 2018, 23, 184 5 of 15
Bothpredicted(Pred.)aswellasexperimental(Exp.)massesaregiveninkDawithexperimentalerrors
loefs sththea inn0d.i0v1i%du.aFlo rsucbalucnuiltast i(oTnaobflet h1e).p Trehdeiscete mdansasteivse smhoawss etsh,aitt haalls obfe etnhet avkaerniainnttso aecxcisotu onft tihdaetnetiaccahl
ssuubbuunniittsc.o ntainsanon-covalentlyboundFADcofactor(molecularmass786Da).
The experimental masses of native and denatured ΔABC do not correspond with the predicted
masses. The observed species appears to be C-terminaNllayt itvruencated [18]. Based on the estimateDde mnaatussr,e d
cleavage occurs before Arg288T,e lteraamdienrg to the removaDl oimf ae rpart of the C-teMrmoninoaml etaril with sequence
(288-RRIAERPENLLLVLRSLVSGLE-309). This truncated form has a predicted subunit mass of
Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp.
71884.9 Da, close to the measured mass of 71899.3 Da [18].
MBP-TtProDHEE* 317.8 319.6 158.9 158.9 79.5 - 78.7 78.7
MBP-TtProDH∆A 310.1 311.3 155.0 154.9–155.7 77.5 - 76.7 76.8
MBP-TTatPbrloe D1.H M∆oAleBcular ma3s0s5e.s1 of E3E0,7 Δ.6A–3, 0Δ7A.8B, and1 Δ52A.6BC as 1d5e2t.e6r–m1i5n3e.7d by 7n6a.t3ive and d-enatured7 E5S.5I- 75.5
MBP-TMtPSr.o BDoHth ∆prAeBdiCcted (Pr3e0d0..)7 as w2e9l0l .a0s– e2x9p0.e7rimen1ta5l0 (.E4xp.) m1a4s5s.0e–s 1a4r6e. 0given7 i5n.2 kDa 7w1i.t9h– e7x2p.7erime7n4t.4al 71.9
errors less than 0.01%. For calculation of the predicted native masses, it has been taken into account
MBP-TtPtrhoaDt eHac∆hA sBubVu3n2itD conta3i0n5s. 2a no2n9-c6o.2v–a3le0n4.t4ly bou1n5d2 F.6AD co1fa4c6t.o5–r 1(m49o.0lecula7r6 .m3ass 77836.0 D–7a6).. 0 75.5 73.0
75.5
MBP-TtProDH∆ABY35F 305.1 307.4 152.5 152.3–152.9 76.3 - 75.5 75.5
Native Denatured
MBP-TtProDH∆ABV36D 305.2 Te2t9ra6m.2er 152.6 Dim15e1r.1–151.9 7M6.3onomer - 75 .5 73.0
75.5
Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp.
*Asdeterminedpreviously[19].
MBP-TtProDH EE * 317.8 319.6 158.9 158.9 79.5 - 78.7 78.7
MBP-TtProDH ΔA 310.1 311.3 155.0 154.9–155.7 77.5 - 76.7 76.8
MBP-TtProDH ΔAB 305.1 307.6–307.8 152.6 152.6–153.7 76.3 - 75.5 75.5
TheMexBpPe-TritmProeDntHa lΔmABaCss eso3f00n.a7 tiv2e90a.0n–d29d0.e7 na1t5u0r.e4 d∆14A5.B0–C14d6.o0 no7t5.c2o rre71s.p9–o7n2d.7 wi7t4h.4t he7p1.r9e dicted
73.0
massesM. BTPh-eTtoPrbosDeHrv ΔeAdBs Vp3e2cDie s3a0p5.p2 ear2s96t.2o–3b0e4.4C -t1e5r2m.6i na1l4l6y.5–tr1u49n.0c ate7d6.3[ 18]7.3.0B–a7s6e.0d o7n5.5t he estimated
75.5
mass, cleavage occurs before Arg288, leading to the removal of a part of the C-terminal tail with
MBP-TtProDH ΔAB Y35F 305.1 307.4 152.5 152.3–152.9 76.3 - 75.5 75.5
sequence(288-RRIAERPENLLLVLRSLVSGLE-309). Thistruncatedformhasapredictedsub7u3.n0 itmass
MBP-TtProDH ΔAB V36D 305.2 296.2 152.6 151.1–151.9 76.3 - 75.5
of71,884.9Da,closetothemeasuredmassof71,899.3Da[18]. 75.5
* As determined previously [19].
2.4. CatalyticProperties
2.4. Catalytic Properties
Figure5presentsanoverviewofthesteady-statekineticpropertiesoftheMBP-TtProDHvariants.
Figure 5 presents an overview of the steady-state kinetic properties of the MBP-TtProDH
ThekineticparametersderivedfromtheseexperimentsaresummarizedinTable2. TheprolineK
m
variants. The kinetic parameters derived from these experiments are summarized in Table 2. The
valuesofEE,∆A,∆AB,and∆ABCarecomparable. However, ∆Aand∆ABhaveaslightlyhigher
activiptyrotlhinaen KEm Evawluheisl eof∆ EAE,B ΔCAi,s ΔaAlmB,o asntdi nΔaAcBtiCv ea.reT choemsepadraatbales. uHgogweesvtetrh, aΔtAα aAnda nΔdABα Bhaavree an solitgrhetqlyu ired
higher activity than EE while ΔABC is almost inactive. These data suggest that αA and αB are not
foropretiqmuiarledac ftoirv oitpyt,iamnadl atchtaivtiftyu,r athnedr thdaetl efutirothneor fdheleeltiixonα oCf hiselcirxi tαicCa ilsf corritcicaatla floyrs icsa.talysis.

FigurFeig5u.reS 5te. aStdeyad-syt-astteatek iknineetitcicss ooff EEEE, ,Δ∆AA, Δ,A∆BA, Ban,da nΔdAB∆CA. BThCe. fTithteed ficuttrevdes caurer vreetsriaevreedr efrtormie vneodn-from
non-lliinneeaarr rreeggrreessssiioonn aannaalylysissi sapapplpylinygin tghet hMeiMchiacehliase-Mlise-nMteenn eteqnuaetqiouna. tEioacnh. Edaatcah pdoaintat wpoasin rtetwrieavserde tirni eved
triplicate.
intriplicate.
Since TtProDH has a low but significant proline oxidase activity [1], it was of interest to address
the oxygen reactivity of the N-terminal arm variants. For all of the variants described above,
Molecules2018,23,184 6of15
Table2. KineticparametersoftheMBP-TtProDHvariantsat25◦C,pH7.4,asdeterminedwiththe
Proline:DCPIPoxidoreductaseassay,usingprolineasthevariablesubstrate,andspecificactivities
oftheMBP-TtProDHvariantsasdeterminedbytheproline:O assay,ataconcentrationof100mM
2
proline.EachdatapointfortheProline:DCPIPassaywasretrievedintriplicate;thedatapointsforthe
Proline:O assaywereretrievedinduplicate.TherawkineticdataoftheProline:DCPIPassay,usedto
2
determinetheKmandkcat,isprovidedinTableS1.
Proline:DCPIPAssay Proline:O2Assay
Km(mM) kcat(s−1) kcat/Km(s−1M−1) SpecificActivity(mU/mg)
MBP-TtProDHEE* 68±8 9.8±0.5 146 266±12
MBP-TtProDH∆A 116±5 12.6±0.3 109 405±15
MBP-TtProDH∆AB 60±3 13.6±0.3 229 228±8
MBP-TtProDH∆ABC 114±8 0.7±0.02 6 20±1
MBP-TtProDH∆ABV32D 309±25 3.2±0.2 10 265±4
MBP-TtProDH∆ABY35F 161±29 14.1±1.5 88 388±5
MBP-TtProDH∆ABV36D 189±27 0.3±0.03 1.6 21±1
*Asdeterminedpreviously[19].
SinceTtProDHhasalowbutsignificantprolineoxidaseactivity[1],itwasofinteresttoaddressthe
oxygenreactivityoftheN-terminalarmvariants. Forallofthevariantsdescribedabove,micromolar
concentrations of enzyme were needed to reliable measure consumption of oxygen, and very low
specific activities were observed (Table 2). Nevertheless, these data confirm that removal of the
completeN-terminalarm(∆ABC)alsoimpairstheprolineoxidaseactivityofMBP-TtProDH.
2.5. ReactionwithN-propargylglycine
TtProDHisirreversiblyinactivatedbythesuicideinhibitorN-propargylglycine[10]. Inactivation
involvestheinitialoxidationofN-propargylglycinetoN-propargyliminoglycineandthesubsequent
formationofabicovalentlinkagebetweenflavinN(5)andtheε-aminogroupofLys99(Figure6C).
Lys99islocatedintheloopbetweenβ2andα2andisinvolvedinbindingthecarboxylicmoietyof
proline[1]. UponreactionwithN-propargylglycinetheabsorptionmaximumofTtProDHat450nm
disappears,whilethemaximumaround380nmgraduallyincreases. Furthermore,thepeakat380nm
shiftstolongerwavelengths. Thisbehaviorisindicativeoftheinitialreductionoftheflavinandthe
subsequentformationofthecovalentLys99-FADadduct[8,10].AfterreactionwithN-propargylglycine,
theenzymeislockedinthereducedstate.
ToprobethecatalyticfeaturesoftheMBP-TtProDHvariantsinfurtherdetail,weinvestigated
thereactivityofEEandthearm-truncatedvariantswithN-propargylglycine. Figure6Ashowsthat
allofthevariantsexcept∆ABCformacovalentflavinadduct,andthatthereactionsresultinsimilar
absorptionchanges,asobservedbeforewithTtProDH[10]. However,amorecarefulanalysisofthe
kineticsofthereactionsrevealssignificantdifferences.
ForEE,∆A,and∆AB,flavinreductionandadductformationareclearlyobserved(Figure6A).
Reduction of EE by N-propargylglycine is relatively slow, as evidenced from the time-dependent
decrease in absorption at 450 nm. Flavin reduction is immediately followed by covalent adduct
formationasevidencedfromtheabsorbanceincreasearound380and405nm. Variants∆Aand∆AB
revealanincreasedrateofreductioncomparedtoEE.Thiscorrespondswiththeincreasedk values
cat
forthesevariants(Table2). Thepeakat380nmshowsaredshiftto385nmforEEandto388nm
for∆Aand∆AB.Furthermore,bothfor∆Aand∆AB,theincreaseinabsorbancearound388nmis
morepronounced.
∆ABCshowsneitherflavinreductionnorformationofaflavinadduct(Figure6A).However,
aslowriseinabsorptionwithamaximumat290nmisobservedinthenear-UVregion,pointingtothe
conversionofN-propargylglycinetoN-propargyliminoglycine. ActivitymeasurementswithDCPIP
suggestthatN-propargylglycineisindeedapoorsubstratefor∆ABC.
Molecules2018,23,184 7of15
Activity of the different enzyme variants before and after incubation with 2.5 mM of
N-propargylglycinewasmeasured(Figure6B).ForEE,residualactivityafter90minofincubation
withtheinhibitorisabout13%. For∆Aand∆ABtheactivitylossisevenmorepronounced,ingood
agreement with the spectral results. The residual activity of ∆ABC after 90 min incubation with
N-propargylglycineisextremelylow(Figure6B).
Molecules 2018, 23, 184 7 of 15

FigFuirgeu6re. R6.e Racetaicvtiitvieitsieos fotfh teheN N-te-tremrmininalala ramrmT TtPtPrrooDDHH vvaarriiaannttss wwiitthh NN--pprrooppaarrggyylglglylycicnine.e (.A()A L)eLfte: ft:
absaobrspotriopntiosnp sepcetrcatrlacl hcahnanggeseso off tthhee vvaarriiaannttss uuppoonn inicnucbuabtiaotnio wnitwhi Nth-pNro-ppraorgpyalrgglyyclginlye.c Tinhee. bTlahcek lbinlaec k
lineinidnidcaicteast etshet hsepescptreucmtr ubmefobree ftohree atdhdeitaiodnd oitfi oNn-porfopNar-pgyrolgplayrcginyel.g Slypceicntera. wSepree crtercaorwdeedre art e1c omrdine d
at 1inmterinvailns tefrovr al9s0 fomri9n0. mRiignh.t: Raibgshotr:baanbcseo rbchaanncgeecsh aonf gtehse ofvatrhieanvtsa riuapnotsn uipnocunbianticounb awtiiothn wNi-th
N-pprrooppaarrggyyllggllyycciinnee ffoolllloowweedd aatt 338800, ,440055, ,aanndd 445500 nnmm; ;((BB) )AAcctitvivitiyty oof fththee ddififfefererennt teennzyzmyme evavrairainatns ts
befobereforaen danadf taefrterin icnucbuabtaitoionn wwiitthh NN--pprrooppaarrggyyllgglylyccinine.e .ErrEorrr obrarbs aarrse abraesebda soend thorneet hrerpeleicraetepsl.i c(aCt)e s.
(C)CChheemmicicaal lsstrtuructcuturer eofo NfN-p-rporpoapragryglgyllygclyincein aenadn tdhet hcoevcaolvenatle LnytsL9y9-sF9A9-DFA adDdaudcdt. uct.
2.6. Interactions between Helix αC and Helix α8
2.6. InteractionsbetweenHelixαCandHelixα8
∆AΔBACBiCs pisr opdroudcuedceads aas caa ctaatlayltyictiaclallylyi mimppaairireeddd diimmeerr.. IInn aaddddiittiioonn,, tthhiiss MMBBPP-T-TtPtProroDDHH vavraiarinatn its is
truncated at its C-terminal helix α8. This suggests that helix αC is important for the stabilization of
truncatedatitsC-terminalhelixα8. ThissuggeststhathelixαCisimportantforthestabilizationof
helix α8. The three-dimensional model of the crystal structure of TtProDH suggests that there is a
helixα8. Thethree-dimensionalmodelofthecrystalstructureofTtProDHsuggeststhatthereisa
hydrogen bond between Tyr35 of αC and Glu295 of α8 (Figure 7). Glu295 is part of the sequence that
hydrogenbondbetweenTyr35ofαCandGlu295ofα8(Figure7). Glu295ispartofthesequencethat
is cleaved off in ΔABC. To investigate whether the Tyr-Glu interaction is important for the
iscleavedoffin∆ABC.ToinvestigatewhethertheTyr-Gluinteractionisimportantforthestabilization
stabilization of helix α8, we changed Tyr35 to Phe. In addition, there is a hydrophobic patch between
ofhelixα8,wechangedTyr35toPhe. Inaddition,thereisahydrophobicpatchbetweenhelixαC
helix αC and α8 (Figure 7). To examine the importance of this patch for the stabilization of helix α8,
andα8(Figure7). Toexaminetheimportanceofthispatchforthestabilizationofhelixα8, Val32,
Val32, and Val36 individually were changed into Asp. The single amino acid substitutions were
andinVtarol3d6uicneddi vinid MuBalPly-TwtPerroeDcHha ΔnAgeBd, siinntcoeA thsips. vTahrieansitn igs lfeuallmy ainctoivaec iadndsu fbosrtmitsu stitoanblsew teetrreaminetrrso.d Iun ctehdisi n
way, we only look at the interaction between helix αC and α8 without possible interference of helix
αA and αB.
Molecules2018,23,184 8of15
MBP-TtProDH∆AB,sincethisvariantisfullyactiveandformsstabletetramers. Inthisway,weonly
lookattheinteractionbetweenhelixαCandα8withoutpossibleinterferenceofhelixαAandαB.
Molecules 2018, 23, 184 8 of 15

FFiigguurree7 7..I Inntteerraaccttiioonnss bbeettwweeeenn hheelliixx ααCC aanndd hheelliixx αα88.. TThhee rreessiidduueess iinn hheelliicceess ααCC aanndd αα88a arreec coololorreedd
iinna ah hyyddrroopphhoobbicic( (rreedd))t oton noonn-h-hyyddrroopphhoobbicic( (wwhhititee))g grraaddieienntt,, aaccccoorrddiinngg ttoot htheeh hyyddrroopphhoobbicicitityys sccaalele
iinnttrroodduucceeddb byyE Eiisseennbbeerrgge etta all..[ [2200]].. TThhee rreemmaaiinniinngg ppaarrtt oofft thheec caattaallyyttiiccd doommaaiinn iisss shhoowwnni inng grreeyya anndd
tthheeF FAADDc coofafacctotorri siss shhoowwnni niny yeellloloww..( (AA))I Ioonnp paaiirrT Tyyrr3355( α(αCC))a annddG Glulu229955( (αα88));;( (BB)) HHyyddrroopphhoobbiiccp paattcchh
bbeettwweeeennα αCCa annddα α88 wwiitthh VVaal3l322( (ααCC))a annddV Vaal3l366( α(αCC))i ninddicicaatetedd..D Daattaaa accccoorrddiinnggt toot thheec crryyssttaalls sttrruuccttuurree
ooffT TttPPrrooDDHH( (PPDDBBe enntrtryy2 2GG3377).).
The far-UV CD spectra of V32D, Y35F, and V36D (Figure 8A) are identical to the far-UV CD-
Thefar-UVCDspectraofV32D,Y35F,andV36D(Figure8A)areidenticaltothefar-UVCD-spectra
osfptehcetrNa -otef rtmhei nNa-ltveramriainnatsl v(Fairgiaunrets3 (AFi)g.uTrhee 3vAis)i.b Tlehefl avvisinibaleb sfolarvpitnio anbpsorroppteiortnie psroofptehreti∆esA oBf vthaeri aΔnAtsB
variants show that the low energy absorption bands of V32D and V36D have shifted about 2 nm to
showthatthelowenergyabsorptionbandsofV32DandV36Dhaveshiftedabout2nmtohigher
whaigvheelern wgtahv,ealsencgotmh,p aarse cdotmopthaoresde otof ∆thAoBsea nodf ΔYA35BF a(Fnidg uYr3e5sF3 B(Faingudr8eBs )3.BT haunsd, t8hBe)fl. aTvhiunsa, btshoer pfltaivoinn
parbospoerrptiteiosno fpVro3p2eDrtaiensd oVf V363D2Dre asnemd Vbl3e6tDho rseeseomf∆blAe BthCo(sFei gouf rΔeA3BBC). (Figure 3B).
Size exclusion chromatography of Y35F indicates that substitution of Tyr35 with Phe does not
SizeexclusionchromatographyofY35FindicatesthatsubstitutionofTyr35withPhedoesnot
aaffffeeccttt thheet teettrraammeerriiccn naattuurreeo off ∆ΔAABB ((FFiigguurree 88CC)).. TThheerreeffoorree,, tthhee iinntteerraaccttiioonn bbeettwweeeenn TTyyrr3355a annddG Glluu229955
does not seem critical for stabilization of helix α8. V32D and V36D elute much later from the gel
does not seem critical for stabilization of helix α8. V32D and V36D elute much later from the gel
filtration column than Y35F, suggesting that these variants mainly form dimers (Figure 8C). Native
filtrationcolumnthanY35F,suggestingthatthesevariantsmainlyformdimers(Figure8C).NativeMS
MS of Y35F confirms the presence of tetramers. Furthermore, V32D and V36D are present both as
ofY35Fconfirmsthepresenceoftetramers.Furthermore,V32DandV36Darepresentbothastetramers
tetramers and dimers (Table 1). The mass of denatured Y35F shows the presence of identical subunits,
anddimers(Table1). ThemassofdenaturedY35Fshowsthepresenceofidenticalsubunits,while
while V32D and V36D show two subunit masses, of which one corresponds with the predicted mass
V32DandV36Dshowtwosubunitmasses,ofwhichonecorrespondswiththepredictedmass(Table1).
(Table 1). The differences between the subunit masses suggest that both V32D and V36D are partially
ThedifferencesbetweenthesubunitmassessuggestthatbothV32DandV36Darepartiallyintact
intact and partially cleaved before R288 (predicted mass 73,001.2 Da), indicating flexibility and
andpartiallycleavedbeforeR288(predictedmass73,001.2Da),indicatingflexibilityandinstabilityof
instability of helix α8.
helixα8.
Analysis of the catalytic properties of Y35F confirms that the interaction between Tyr35 and
Glu295 is not crucial for the functioning of TtProDH (Figure 8D). The minor decrease in catalytic
efficiency when compared to ΔAB mainly results from a slight increase in Km for the proline substrate
(Table 2). V32D and V36D, on the other hand, are poorly active (Figure 8D). With these variants, a
considerable decrease in catalytic efficiency is observed (Table 2). Oxidase activity of especially V36D
is very low (Table 2), as found with ΔABC.
Molecules2018,23,184 9of15
Molecules 2018, 23, 184 9 of 15

FiFgiugurere8 .8.P Proroppeertriteiesso offh heelilxixα αCCv vaarriiaannttss ooff ∆ΔAABB.. ((AA)) FFaarr--UUVV CCDD ssppeeccttrraa;; ((BB)) VViissiibbllee ffllaavviinn aabbssoorrppttiioonn
spsepcetcrtar;a(; C(C))H Hyyddroroddyynnaammicicp prrooppeerrttiieessa ass mmoonniittoorreedd bbyy SSuuppeerrddeexx 220000 ssiizzee--eexxcclluussiioonn cchhrroommaatotoggrraapphhyy; ;
anadnd,(, D(D))S tSetaedadyy-s-tsatatetek kinineetitcicd daattaaa assd deetteerrmmiinneedd wwiitthh tthhee PPrroolliinnee::DDCCPPIIPP aassssaayy.. EEaacchh ddaattaa ppooiinntt wwaass
rertertireiveevdedin int rtirpiplilciactaet.e.T Thheer raawwk kinineetticicd daattaao off tthhee PPrroolliinnee::DDCCPPIIPP aassssaayy,, uusseedd ttoo ddeetteerrmmiinnee tthhee KKmm aanndd
kckactatv vaalluueess,, iiss pprroovviiddeedd iinn TTaabbllee SS11..
The catalytic features of the helix αC variants were investigated in more detail by probing their
Analysis of the catalytic properties of Y35F confirms that the interaction between Tyr35 and
reactivity with the suicide inhibitor N-propargylglycine (Figure 9). For Y35F (Figure 9A), the rates for
Glu295 is not crucial for the functioning of TtProDH (Figure 8D). The minor decrease in catalytic
flavin reduction and adduct formation are similar as found for ΔAB (Figure 6A). For V32D, flavin
efficiencywhencomparedto∆ABmainlyresultsfromaslightincreaseinK fortheprolinesubstrate
reduction and adduct formation are also observed (Figure 9A), but these pmrocesses are much slower
(Table 2). V32D and V36D, on the other hand, are poorly active (Figure 8D). With these variants,
than for ΔAB. The flavin prosthetic group of V36D is not reduced by N-propargylglycine, and the
aconsiderabledecreaseincatalyticefficiencyisobserved(Table2). OxidaseactivityofespeciallyV36D
typical absorption increase around 380 nm, indicative for adduct formation, is also not observed
isverylow(Table2),asfoundwith∆ABC.
(Figure 9A). However, as for ΔABC, a slow rise in absorption occurs in the region 300–350 nm,
ThecatalyticfeaturesofthehelixαCvariantswereinvestigatedinmoredetailbyprobingtheir
pointing to the conversion of N-propargylglycine to N-propargyliminoglycine.
reactivitywiththesuicideinhibitorN-propargylglycine(Figure9). ForY35F(Figure9A),therates
All the helix αC variants lose activity when treated with N-propargylglycine. After incubation
for flavin reduction and adduct formation are similar as found for ∆AB (Figure 6A). For V32D,
for 90 min, Y35F is almost completely inactivated, but V32D and V36D retain 30% and 63% of their
flavinreductionandadductformationarealsoobserved(Figure9A),buttheseprocessesaremuch
original (rather low) activity (Figure 9B).
slowerthanfor∆AB.TheflavinprostheticgroupofV36DisnotreducedbyN-propargylglycine,and
thetypicalabsorptionincreasearound380nm,indicativeforadductformation,isalsonotobserved
(Figure9A).However,asfor∆ABC,aslowriseinabsorptionoccursintheregion300–350nm,pointing
totheconversionofN-propargylglycinetoN-propargyliminoglycine.
AllthehelixαCvariantsloseactivitywhentreatedwithN-propargylglycine. Afterincubation
for90min,Y35Fisalmostcompletelyinactivated,butV32DandV36Dretain30%and63%oftheir
original(ratherlow)activity(Figure9B).
MolMecuolleecsu2l0es1 82,02138,, 12834, 184 10o1f01 o5f 15

FigFuirgeu9r.e R9.e aRcetiavcittiiveistioesf hoef lihxelαixC αvCar ivaanrtisanotfs ∆oAf BΔAwBit hwNith-p Nro-pparrogpyalrgglyylcginlyec.in(eA. )(LAe)f tL:eaftb: saobrpsotiropntion
spescptreacltrcahla cnhgaensgoefs tohfe tvhaer viaanrtisanutps ounpionnc uinbcautiboantiwonit whiNth-p Nro-ppraorpgayrlggylylgcilnyec.inTeh. eThblea cbklalcikn eliinned iincadtiecsates
thetshpee scptreucmtrubmef boreefotrhee tahded aidtidointioonf Nof- pNro-ppraorpgayrlggylylgcilnyec.inSep. eScptreactwrae wreerreec orercdoerddaedt1 amt 1i nmiinnt einrvtearlvsafolsr for
90m90i nm.iRni.g Rhitg:hatb: saobrsboarnbcaencchea cnhgaensgoefs tohfe thvea rviaanritasnutsp ounpoinnc iunbcautbioantiowni twhiNth- pNr-oppraorpgayrlggylylgcilnyecifnoel lfoowlloedwed
at3a8t0 3,8400,5 4,0a5n,d an4d50 4n5m0 n;m(B;) (AB)c tAivcittiyviotyf tohfe thdeif fdeirfefenrteenntz eynmzyemvaer viaanritasnbtesf boerefoarned anafdt earftienrc uinbcautbioantiownit whith
N-pNro-ppraorpgayrlggylylgcilnyec.inEer.r Eorrrboarr bsaarrse abraes bedasoend tohnr etherreeep lriecpaltiecsa.tes.
3. D3.i sDciusscsuisosnion
ProPlirnoelidneeh dyedhryodgerongaseensacsoens tcaoinntaacino nas ecrovnesder(vβeαd) 8(βTαIM)8 -TbaIMrre-bldarormela dinoamnadina nanNd-t earnm Nin-atelramrmintahl aatrm
difftehrast indilfefnegrsth inam leonnggthm oanmoofunngc tmioonnaolfbuancctetiroianlaaln bdaecutekraiaryl oatnicdP erouDkaHrsy.oItnict hPirsosDtuHdsy., wIne itnhvise ssttiugdatye,d we
theinfuvnecsttiiognaateldim tphaec tfuofntchtieoNna-tle rimmpinaaclt aromf tohfeT hNer-mteurmstihnearlm oaprmhil uosfP TrohDerHm.uWs ethaenramlyozpehdiluvsa rPiarnotDsHth.a tWe
lackanoanlyez(e∆dA v),atrwiaont(s∆ tAhBat) ,laocrkt horneee ((Δ∆AA)B, Ctw)No -(tΔeArmBi)n, aolrh tehlriecee s(ΔanAdBcCo)m Np-aterremditnhaels ehevlaicreiasn atnsdto ctohmepEaEred
vartihaensteo vfaMriBanPt-sf utose tdheT tEPEro vDaHria.nTth oefl MattBePr-vfuarsieadn tTitsParohDigHh.l yThaec tliavtetesro vluabrilaenfto rism ao hfitghhelye nazcytimvee stohlautble
excfloursmiv oelfy thfoe remnzsytmetrea tmhaetr sex[1cl9u].siSvpeelyc tfroarlmans atelytrsaismsehros w[1e9d]. tShpaetcttrruanl acantailoynsiosf sthhoewNe-dte trhmati ntraulnacramtioonf of
TtPtrhoeD NH-tdeormesinnaeilt haerrma fofefc tTtthPerobDinHd indgoeosf tnheeitFhAeDr acofffeacctt otrhne obritnhdeinmgi corof etnhvei roFnAmDe nctoofafctthoer flnaovri nthe
isomalliocrxoaezninveirroinnmg.ent of the flavin isoalloxazine ring.
WeWaler eaalrdeyadshyo swhoewdethda tthnaot nn-onna-tnivaetivage gargeggraetgioantioonf MofB MP-BTPt-PTrtoPDroHDiHsd isu edutoe tthoe thhey dhryodprhoopbhiocbitiycity
ofhofe lhixelαixA α.ARe. pRleapcliancginPgh eP1h0e1a0n danLde uL1e2u1o2f αofA αwAi twhigthlu gtalumtaamteasteelsi meliinmaitneasteths ethfoer fmoramtioantioonf loafr glaerrger
aggargeggraetgesat[e1s9 ][.1H9]e. rHe,ewree, ewstea belsisthabedlisthheadtt htheacto tmhep lceotemrepmleotev arleomfohvealilx oαf Ahe(∆liAx )α,Aor r(ΔemAo),v oalr orfehmeolivceasl of
αAhaenlidcesα BαA(∆ AanBd), hαaBs (tΔhAeBsa),m heaesf ftehcet :snaomaeg egfrfeegcat:t ensoa raeggorbesgearvteesd aarned o∆bsAeravnedd ∆aAndB ΔexAc luasnidv eΔlyAB
foremxctleutsriavmeleyr sf.orTmh itsectroanmfierrms.s Tthhiast cαoAnfiirsmress tphoant sαibAl eisf orerstphoenisnibvliet rfoora tghger eing avtiitorno aogfgMreBgPa-tTiotnP roofD MHBP-
andTttPhraotDbHo thanhde litcheast αbAothan dheαliBcesa reαAn oatnedss eαnBti aalrfeo rnotht eetsesternatmiael rifzoart itohne ptreotrcaemsse.riFzuartitohne rmproorcee,ss.
estFimuratthioenrmoforkei,n eetsitcimpaartaiomne otefr skirneevteiac lepdartahmateαteArsa rnedveαaBleadr ethnaott eαsAse nantidal αfoBr tahree ennozty emssaetinctiaaclt ifvoirt ythe
ofMenBzPym-TatPtirco aDcHtiv.iAtyc touf aMllyB,P∆-TAtBPriosDthHe. mAcotsutaalclyti,v ΔeAMBB iPs -tfhues emdoTstt ParcotiDvHe MvaBrPia-fnutsreedp TotrPterdoDthHu svafarira.nt
Incruebpaotriotends wthiuths thfaer.m Iencchuabnaistimon-bsa sweidthin thhieb itmorecNh-apnriospma-rbgayslgedly ciinnheibyiiteoldr eNd-spurpoppoarrtgiynlggliyncfoinrme aytiieolnded
abosuutppthoertcinatga liyntfiocrmcoamtiponet eanboceuta nthde tchaetasltyrtuicc tcuormalpientteengcreit aynodf t∆hAe satrnudct∆uAraBl .inTtheegrflitayv oinf ΔcoAfa acntodr ΔoAf B.
The flavin cofactor of both arm-truncated variants is rapidly reduced, immediately followed by
covalent adduct formation. This leads to irreversible suicide inactivation of the enzymes by N-
propargylglycine (Figure 6).