Mechanism and Regulation Mg

8/18/2019 Mechanism and Regulation Mg

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Biochem. J. (1997) 327, 321±333(Printed in Great Britain) 321REVIEW ARTICLE

Mechanism and regulation ofMgchelataseCaroline J. WAL!"#1 and "o$ert %.W&LL'W.#%eartment o* Biolo+ical cience,Clemon -nierit/, Clemon, C 2903193, -..A., and .%iiion o* Biolo+/and 4edicine, Bro5n -nierit/,Proidence,"& 2912, -..A.4+chelatae catal/e the inertion o*4+ into rotoorh/rin

&6 (Proto). hi eemin+l/ imlereaction alo i otentiall/ oneo* the mot interetin+ and cr8cial tein the ($acterio)chloroh/ll (BchlChl)/ntheiath5a/, o5in+ to it oition

at the $ranchoint $et5een haem andBchlChl /nthei. -8ntil the leel o* Proto, haem andBchlChl /nthei hare acommon ath5a/. :o5eer, at the ointo* metalion inertionthere are t5o choice; 4+e BchlChl


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(catal/ed $/ 4+chelatae) or ?ee haem (catal/ed$/ *errochelatae). h8 the relatie

actiitie o* 4+chelataeand *errochelatae m8t $e re+8lated5ith reect tothe or+anim@ re8irement *or theeend rod8ct . :o5 i thiMgC!ELATA"E# A $RA%C!&'I%T

E%()ME!n/me at the $ranchoint o*$io/nthetic ath5a/ haeal5a/ $een o* interet to $iochemit$eca8e, a a +eneral r8le,the/ are ti+htl/ re+8lated accordin+ tothe reailin+ meta$oliccondition. &n hoto/nthetic or+anim,a lar+e art o* their$io/nthetic eort i 8t into the/nthei o* chloroh/ll (Chl)or $acteriochloroh/ll (Bchl). :o5eer,BchlChl /nthei

hare a common $io/nthetic ath5a/5ith a econd endMg-chelatase ALA Protoporphyrin IXMg-protoporphyrin IXOther chlorophylls

Chlorophylla

Phytochromobilin


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&n all hoto/nthetic or+anim t8diedto date, 4+chelatae ia threecomonent en/me, and in

eeral ecie thee roteinhae $een cloned and eKreed in anactie *orm. he reactionta>e lace in t5o te, 5ith an APdeendent actiation*ollo5ed $/ an APdeendent chelation

te. he actiation tema/ $e the >e/ to re+8lation, altho8+hariation in 8$8nitleel d8rin+ di8rnal +ro5th ma/ alola/ a role in determinin+the 8K thro8+h the BchlChl and haem$ranche o* the ath5a/.rod8ct, haem (cheme 1). ince haemand BchlChl are eachneeded in ario8 amo8nt accordin+ todeelomental ta+eandor li+ht condition, the coordination o* haem and BchlChl

/nthei ha $een an area o* ecialinteret. he $ranchoint o* haem and BchlChl/nthei i at theleel o* rotoorh/rin &6 (Proto)8tiliation *or metalion


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chelation. &n haem /nthei, a *erro8ion i inerted into thetetra/rrole rin+ $/ the en/me

*errochelatae. &n BchlChl/nthei, an 4+ ion i inerted intoProto $/ 4+chelatae322 C. J. Wal>er and ". %. Willo5MgMg-chelatase

N NN NCO O!CO O!CO O!CO O!N N

N NMg-A"PProtoporphyrin IX Mg-Protoporphyrin IX"cheme 2 Reaction catal-sed *- Mgchelatase(cheme 2). Characteriation o* theroertie o* the t5o $ranchoint


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chelatae ho8ld thro5 li+ht on ho5 theleel o* haemand BchlChl are coordinated in

hoto/nthetic cell.?errochelatae ha $een characteried in$oth hoto/ntheticand nonhoto/nthetic or+anim; it ia mem$raneaociatedrotein o* a$o8t >%a. Catal/i $/ thi

en/me aear to $etrai+ht*or5ard, re8irin+ onl/ Proto and*erro8 iron a 8$trateE1F. &n contrat, 4+chelatae hareited attemt atcharacteriation *or man/ /ear and haroed to $e a m8chmore diMc8lt en/me to anal/e. hemaNor imediment 5a thelac> o* an in .itro aa/, 5hich 5a onl/achieed 0 /ear a+o E2F.ince that time o8r >no5led+e o* 4+chelatae ha eKanded

dramaticall/ to the eKtent that theen/me ha $een cloned ande8enced *rom eeral hoto/ntheticecie E3±1F. he 8roe o* thi "eie5 i to $rin+to+ether the in*ormation


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on 4+chelatae that ha $een 8$lihedince the reie5 $/Beale and Weintein in 199 E11F, in

5hich the >no5led+e o* 4+chelataeat that time 5a decri$ed in +reatdetail. With aolo+ieand ac>no5led+ment to thoe a8thor5ho did the ioneerin+t8die on 4+chelatae, thi reie5 5ill

onl/ to8ch $rie/ 8ontheir earl/ 5or>. he rt hal* o* thireie5 5ill concentrate onthe en/molo+/, tr8ct8re andmechanim o* 4+chelataeD theecond hal* 5ill dic8 h/iolo+icalaect 8ch a localiationo* the en/me and the re+8lation o* the$ranchoint 5ithreect to $oth en/me and +enere+8lation..EM'%"TRATI'% '/ MgC!ELATA"E ACTIVIT) I% VITR'

&n the 197 and earl/ 19O, attemt5ere $ein+ made to aa/4+chelatae actiit/ in $othhoto/nthetic $acteria and hi+herlant. &n $oth /tem, reearcher*o8nd t5o *eat8re in


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common. ?irt, actiit/ co8ld $emea8red in chlorolat (hi+herlant) or 5hole cell ($acteria), $8t

$rea>a+e o* the chlorolator cell inaria$l/ re8lted in either acomlete lo or aroK.9O lo o* actiit/ E12±17F. econdl/,in thee /tem, 4+chelation5a onl/ o$ered in the reence o*

AP E13,1F. he reaon *or the lo o* actiit/ on$rea>a+e $ecame aarent5hen hi+h in .itro leel o* 4+chelataeactiit/ 5ere reorted*or l/ed ea (&isum sati.um)chlorolat E2F. he >e/ toreerin+ actiit/ 5a to l/echlorolat at a hi+h roteinconcentration o a not to dil8te thechlorolat content. Whenthi l/ed rearation 5a earatedinto a tromal and a

mem$rane *raction, the actiit/ 5a lot.:o5eer, recom$inationo* thee t5o *raction retored actiit/. he a8thorconcl8ded that 4+chelatae m8t $e am8lticomonent en/me,


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re8irin+ at leat t5o rotein, onemem$raneaociated andone ol8$le. &nteretin+l/, the actiit/

5a till a$ol8tel/ deendenton AP, eliminatin+ an/ oi$ilit/ thatthe APre8irement 5a an arti*act o* aa/in+the en/me in 5holechlorolat or haerolat.

&t i 5orth notin+ that, initiall/, theere8lt 5ere conidered8rriin+. ince *errochelatae and 4+chelatae carr/ o8tirt8all/ identical reaction, there 5athe a8mtion that theeen/me 5o8ld $e imilar (i.e. a in+lemem$rane$o8nd rotein).hortl/ a*ter the reort o* in .itro actiit/*rom ea 5a8$lihed, the mea8rement o* hi+h in.itro 4+chelatae actiit/*rom l/ed c8c8m$er (Cucumis sati.us)

etiolat 5a decri$edin 5hich actiit/ 5a conned to themem$rane *raction E1OF. &nthat t8d/ the a8thor +entl/ l/ed theetiolat in a *ortiedmedi8m containin+ hi+h concentration

o* 4+Cl


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Proto. &nteretin+l/, the addition o*ol8$le rotein $ac> to themem$rane had a li+ht inhi$itor/ eect

on 4+chelatae actiit/,rein*orcin+ the a8thor@ concl8ion that4+chelatae 5a amem$rane$o8nd en/me.4+chelatae ha no5 $een conrmedto $e a m8lticomonent

en/me in eeral or+anim $/ $oth$iochemical aa/ andmolec8lar +enetic E,0,19F. While theconcl8ion o* Lee et al.E1OF ma/ initiall/ eem inalid, morerecent data ha 8++etedthat 4+chelatae *8nction a am8lticomonent comleKE2,21F. &* thi roe to $e the cae, Leeet al.@ data can $ereinterreted to 8++et that thereence o* hi+h concentrationo* Proto, AP and 4+


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E23F. &n theor/, the en/me 5hich8tilie thi intermediate a a8$trate can $e ai+ned to the

m8tated +ene. he three +ene, *chI, *ch. and *ch!,5ere eKected to code*or 4+chelatae 8$8nit, a the 4+chelatae rec8ror, Proto,acc8m8lated 5hen the/ 5ere each

dir8ted E2F. Gi$on et al.conrmed thi ai+nment$iochemicall/, $/ clonin+ thee +eneinto eKreion ector EF. heeKreed rotein 5ere allol8$le, and 5hen com$ined had 4+chelatae actiit/. hea8thor *o8nd that the/ onl/ o$ered4+chelatae actiit/5hen all three rotein 5ere reent. he leel o* actiit/ 5erehi+h (2 nmolh er m+ o* rotein), anda+ain, the actiit/

re8ired AP. he *ch. +ene i *o8ndimmediatel/ a*ter the *chI+ene a art o* the ame oeron in thehoto/nthei +enecl8ter, and the a8thor *o8nd that the/5ere onl/ a$le to +et


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8$8nit *raction that 5ere all re8ired*or 4+chelatae actiit/,roidin+ conincin+ eidence that the

en/me ha three 8$8nit.A imilar re8lt ha $een o$tained *romthe c/ano$acteri8m"-nechoc-stis E0F 5here, on the $ai o*e8ence imilaritie tothe Rhodo*acter 4+chelatae +ene,

three 8tatie 4+chelatae+ene 5ere iolated and cloned. ince"-nechoc-stis i a Chlratherthan a Bchlcontainin+ or+anim, theethree +ene 5eredenoted chlI, chl! and chl. E0,7F.!Kreion clone 5erecontr8cted *or all three 8$8nit, 5iththe & and % 8$8nit inearate ector. All three eKreedrotein 5ere ol8$le andall 5ere necear/ *or 4+chelataeactiit/.

/RACTI'%ATI'% '/ MgC!ELATA"E I%!I0!ER&LA%T ETRACT"Are there alo three rotein *or 4+chelatae in hi+her lantIAltho8+h there ha $een le 8cce

5ith iolatin+ 4+chelatae


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+ene *rom hi+her lant (otential 4+chelatae +ene that hae$een iolated o *ar 5ill $e dic8ed in

detail $elo5), there ieidence that 4+chelatae alo ha atleat three 8$8nit inthee or+anim. hi eidence come*rom m8tant t8die androtein *ractionation 5or>.

&n $arle/ (!ordeum .ulgare), there i aerie o* Chl/ntheim8tant 5hich hae $een identied onthe $ai o* the acc8m8lationo* intermediate 5hen *ed the Chlrec8ror aminolae8linic acid (ALA). hree nonallelic m8tant, anthaf , anthag and anthah,acc8m8late Proto 8nder thee conditionE2,20F. &n deeloin+chlorolat iolated *rom theem8tant, no 4+chelatae actiit/

5a detecta$le E7F. :o5eer, theair5ie com$ination o* l/ed chlorolat eKtract *rom theem8tant retored actiit/E19F. he ded8ction *rom thee aa/ ithat each m8tant ha a


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5ill alo hae a threecomonenten/me.MgC!ELATA"E MTA%T"

A dic8ed a$oe, BchlChldecientm8tant o* hoto/ntheticor+anim hae $een inal8a$le in theidentication o* the tr8ct8ral +ene reoni$le *or 4+chelatae. hee 4+chelatae

m8tant hae +enerall/ $een identied$/ their ina$ilit/to ma>e BchlChl and $/ theiracc8m8lation o* the en/me@8$trate, Proto. eeral eKamle o*4+chelatae m8tant aredecri$ed in thi "eie5, and *or theem8tant it ha $eenoi$le to identi*/ 5hich o* the three8$8nit i m8tated.:o5eer, there are alo eeral otherm8tant 5hich ati*/ theecriteria and hae $een 8++eted in the

literat8re to $e 4+chelataem8tant. &t ho8ld $e noted that thee8tatie4+chelatae m8tant hae not $eenconrmed either $/ $iochemicalaa/ or $/ the e8ence o* the

m8tated +ene, and the


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imortance o* 8ch additionalidentication cannot $e oertated.?or eKamle, m8tation in

rotoorh/rino+en oKidaealo dila/ the henot/e o*acc8m8latin+ Proto, d8e to theoKidation o* the acc8m8latedrotoorh/rino+en E29F. Additionall/,it ha $een noted that the *rs2 m8tant

o* Chlam-domonasi de*ectie in chlorolatmem$ranedeelomentand ha a leiotroic eect that ca8ethe acc8m8lation o* ProtoE3F D thee a8thor alo noted thatome carotenoid /ntheim8tant alo acc8m8late ome Proto.?or the a>e o* comletene, and a a+eneral re*erence, a$le1 lit all o* the 4+chelatae m8tant,$oth 8tatie andconrmed. &n each cae the reaon *or

claication i +ien and5here oi$le, the m8tant 8$8nit iidentied. With the c8rrentaaila$ilit/ o* ro$e and anti$odie it ioi$le that ome o* thee 8tatie m8tant 5ill $e identied

in the near *8t8re.


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MEC!A%I"M '/ MgC!ELATA"E he rt i+nicant ro+re to5ard8ndertandin+ the mechanim

o* 4+chelatae came *rom thedeeloment o* a contin8o88orimetric aa/ in 5hich chlorolat*raction 5ith alo5 Chl content 5ere 8ed E31F. With theimroed time reol8tion

o* thi aa/, it $ecame aarent thatthere 5a a la+ o* eeralmin8te $e*ore 4+chelatae actiit/5a o$ered a*ter theaddition o* all the 8$trate. hi la+ inactiit/ co8ld $eeliminated i* the en/me 5areinc8$ated 5ith AP, 5hich8++eted that there i a t5ota+eroce; an APdeendentactiation *ollo5ed $/ 4+chelation. here*ore, the *8rtherdic8ion o* the mechanim o* 4+

chelatae 5ill $e decri$ed interm o* the Q actiation@ and [email protected] C. J. Wal>er and ". %. Willo5Ta*le 1 $chl,Chlde4cient mutants of +hotos-nthetic *acteria5 algae and

+lants 6ith the demonstrated or


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+ro*a*le defect at the Mgchelataseste+ hee m8tant acc8m8late Proto either

8nder normal +ro5th condition or 5hen*ed ALA.ecie 48tant 8$8nit eectedComment "e*erenceR ca+sulatus RS0 : &nteroon m8tanto* *ch! E73F

%B01 % &nteroon m8tant o* *ch. E23F%B3 & &nteroon m8tant o* *chI E23FR s+haeroides %01 % otal o* ninetranoon m8tant o* *ch. ome o*5hich ma/ $e in *chI E0,7F& & ranoon m8tant o* *chI E7F:3, :33, :21 : ranoonm8tant o* *ch!D total o* 2 : m8tantE0,7F! 8ulgare ($arle/) 9anthaf : 9anthaf2: i lea>/ D other allele are comletel/$loc>ed E7,19,20,7F 9anthag % 9anthag; i lea>/ D other

allele are comletel/ $loc>edE7,19,20,7F 9anthah & All m8tant allele arecomletel/ $loc>ed E7,19,20,7Fchlorina1no5n 4a/ $e re+8lator/m8tant E77F


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(ea ma-s (maie) oil -ello6 1 -n>no5no-1ed D o-1/ D non allellic to l13 or l>$land-

E7OFl>$land- -n>no5n Comletel/$loc>ed D non allellic 5ith o-1 or l13 E7OFl13 -n>no5n Lea>/ m8tant D nonallellic5ith o-1 or l>$land- E7OFTriticum .(5heat) chlorina21, C%3,

chlorina1 -n>no5n All m8tant lea>/ Dred8ced amo8nt o* chloroh/ll * E77F Antirrhinum ma?us oli8e : ranoonta++ed chl! EOF A thaliana chlorata2 & %TAta++edchlI E9F 9antha2 -n>no5n Tonallelic to 9antha3 E79F 9antha3 -n>no5n Tonallelic to 9antha2 E79FChlorella 8ulgaris WB -n>no5n EOFChlam-domonas reinhardtii *rs2-n>no5n Lethal 5hen +ro5n in li+ht D

ro$a$l/ de*ectie in latid mem$ranedeeloment E3,O1F*rs1 -n>no5n Lethal 5hen +ro5n inli+ht D trace o* Chl in the dar> D nonallelic to *rs2 or *rc1 E3,O1F


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*rc1 -n>no5n Tormal henot/e inli+ht D trace o* Chl in dar> and Protoacc8m8late D non allelic E3,O1F

to *rs2 or *rs1 -- -n>no5n 4a/ $e leiotrohic m8tantanalo+o8 to *rs2 EO2F&n ea chlorolat eKtract, eitheradenoine HEcthioF PTtrihohate (APEF) or AP co8ld

oercome the la+ eriod,iml/in+ that either 8$trate co8ld $e8tilied *or actiationE2F. APEF i t/icall/ 8ed a a rotein>inae 8$trate,raiin+ the oi$ilit/ that roteinhohor/lation ma/ la/ aart in the actiation roce. :o5eer,5hen APEF 5a 8ed*or actiation, the 8$e8ent addition o* AP 5a re8ired *or4+e lace. hiimlied that AP 5a alo

re8ired *or the 4+chelation te andthat the role o* AP ma/$e dierent in each o* thee t5o te. he otimal APconcentration *or $oth te 5adetermined and *o8nd to $e


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onl/ re8ired *or the chelation te. hire8lt ha recentl/ $eenconrmed in *ractionated ea

chlorolat, 5here the *ractiono* the hi+herlant & and % co8nterartare re8ired *oractiation E2OF. he role o* the &, % and : rotein canonl/ $e +8eed at.

When the : rotein i eKreed in hotcell, it ha Proto noncoalentl/$o8nd E,21F. &n the 8ried rotein, theProto:8$8nit ratio i 1;1 E21F. %/namicli+htcatterin+ t8die haeho5n that 8ried : i reent a amonomer, 5herea & ireent a a dimer. ?or otimal actiit/ aratio o* one & dimer toone : monomer i re8ired E21F. helac> o* 8re % rotein didnot ena$le thi t8d/ to $e eKtended to

all 8$8nit, altho8+h it5o8ld eem that % i onl/ needed iner/ mall amo8nt E0,21F.C!EMICAL C'%"I.ERATI'%" /'R Mg2=I%"ERTI'%&t i oi$le to +ain an ini+ht into the

mechanim o* an en/me


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$/ eKaminin+ ho5 the reaction ta>elace in the a$ence o* $iolo+ical catal/t. he chemitr/ o* the

metalion inertion intoorh/rin, termed Qmetalation@, ina8eo8 and nona8eo8/tem ha $een eKteniel/ reie5edE32,33F, o the dic8ion5ill $e limited to aect 5hich $ear

directl/ on 4+chelatae. he mechanim o* metalation o*orh/rin 5ith $ialentmetal ion re8ire at leat *o8r te5hich ma/ occ8r e8entiall/or in concert; (&) derotonation o* the/rrole nitro+enatomD (&&) remoal o* li+and that arecoordinated to the metalion or metalion carrier, *or eKamle,5ater molec8leD (&&&) coordinationo* the metal ion 5ith the *o8r /rrolenitro+en atomD

(&U) comletion o* the coordinationhere, i* re8ired, 5ith anaKial li+and E32F. he eae 5ith 5hich ametal i inerted into aorh/rin 5ill deend in art on all o*thee arameter. ?or


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eKamle, C8cal)mol) *or the

catal/ed reaction.Gien the ener+etic adanta+e o* 8in+the catal/ed reaction, it4echanim and re+8lation o* 4+chelatae 32;H

H


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H

/igure 1 Consensus seuence deri8edfrom the deduced amino acid seuences

of the !su*unit genes he conen8 5a created *rom anali+nment o* the : 8$8nit *rom Ara*ido+sis thaliana, !ordeum 8ulgare, Antirrhinum ma?us, "-nechoc-stis P.C.C.0O3 and Rhodo*acter ca+sulatus 5ith

GenBan> acceion n8m$er R0O9,-20910, 6731, -29131 and 471reectiel/. Q # @ &ndicate comletel/conered reid8e and Q @ indicateconeratie 8$tit8tion. Lo5ercae QK @indicate amino acid that are notconered. he n8m$erin+ i *or the Ara*ido+sis seuence and tart *romoition 1, 5hich i a*ter the chlorolattranit e8ence. Ga created $/ ile8are indicated $/ Q@. "eid8e that are

8nderlined are conered in the other :8$8nit co8nterart D the CochelataeCo$T *rom &seudomonas denitri4cans,acceion n8m$er 402O00D the8tatie co$altochelatae and nic>lechelatae homolo+8e *rom

Methanococcus ?annaschii, acceion


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n8m$er -07O and -073, and"-nechoc-stis PCC 0O3, acceionn8m$er %99.

he three conered hitidine reid8eare colo8red red. he ro+ram in theGCG ro+ram ac>a+e (GCG &nc.,4adion, W&, -..A.) 5ere 8ed to helcreate thi dia+ram.i ro$a$le that the $iolo+ical inertion

o* 4+


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4+chelatae thi $ae 5o8ld aloro$a$l/ $e a hitidine reid8eand 5hen the en/me releae the 4+

rotoorh/rin(4+Proto), the *th li+and on the roteinma/ $e relaced $/ a5ater molec8le.o 5hat i the *8nction o* AP in the 4+chelatae reactionI

A cl8e to the role o* AP ma/ $e *o8nd inthe relatie rate o* $ialentmetalion inertion intoorh/rin, 5hich *ollo5 theorder E33,3F ;C8VRnV4n, Co, ?eVTiVCd(4+ he a$oe rate correlate 5ith the a$ilit/o* thee metal ion torender la$ile a coordinated 5atermolec8le, and the oition o* 4+


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the 4+


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eidence that the 4+


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conered moti*, ec8late a to the*8nction o* the 8$8nit. here are c8rrentl/ 13 co8nterart o*

the & 8$8nit o* 4+chelataein the GenBan> T8cleotide e8ence%ata$ae thathae at leat 9, and 8 to O0,identit/ 5ith each other at theamino acid leel. hee rotein ran+e in

ie *rom 37 >%a *orthe chlorolatencoded 8$8nit & o*'listhodiscus luteus(a 8nicell8lar al+a) to 0 >%a *or then8clearencoded Ara*ido+sisthaliana (thale cre) 8$8nit &co8nterart 5hich incl8de a Tterminalchlorolat tranit etide EF. &t iinteretin+ that onl/in the cae o* the nonchloroh/teal+ae are the 8$8nit &e8ence encoded $/ the chlorolat+enome. An ali+nment o*

thee e8ence 5a 8ed to create theconen8 e8ence ho5nin ?i+8re 2. &t i note5orth/ that theimilarit/ dendo+ram that5a +enerated $/ the GeneticCom8ter Gro8 (GCG) ro+ram


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Qile8@ 5hen thee e8ence 5ereali+ned 5a imilar to theh/lo+enetic relationhi o* thee

or+anim ded8ced $/ acomarion o* tufA +ene E30F.All o* the 8$8nit & e8ence hae thethree 4+APaemoti* (?i+8re 2) identied $/ oonin,and $/ thi +ro8in+ the

& 8$8nit e8ence are in a 8er*amil/o* 8tatie %TAdeendentAPae E37,3OF. he & 8$8nite8ence can $e*8rther +ro8ed to a 8$*amil/ 5hoemem$er are inoled inthe initiation o* %TA relication or intrancrition re+8lation.:o5eer, ince there i no eKerimentaleidence to indicate that4+chelatae re8ire %TA *or actiit/, iti mot ro$a$le thatthe e8ence imilarit/ i d8e olel/ to

tr8ct8ral and *8nctionalconeration o* the APae domain inthi 8$8nit. &t i eKectedthat one o* the 4+chelatae 5ill haean APae actiit/, inceAP h/drol/i i re8ired *or actiit/. &t

there*ore eem


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reaona$le to redict that the & 8$8nit$ind to, and h/drol/e,AP d8rin+ the reaction.

% 8$8nit e8ence are le a$8ndantin the GenBan>%ata$ae, 5ith onl/ *o8r co8nterart o*thi 8$8nit. 'nl/ threeo* thee e8ence encode *or 4+chelatae 8$8nit, 5ith the

*o8rth $ein+ a 8tatie Tichelatae8$8nit. he % 8$8nithae calc8lated molec8lar mae o* O7,73 and 0 >%a *or theVD E L N

G K

/igure 2 Consensus of I su*unitssho6ing the three conser8ed AT&ase*inding motifs 37D he e8ence n8m$erin+ i *or the Ara*ido+sis thaliana e8ence and tart*rom oition O0, 5hich i a*ter thechlorolat tranit e8ence. he

conen8 i *rom an ali+nment o* the &8$8nit*rom Cr-+tomonas +hi5 Methanococcus ?annaschii5 Ara*ido+sis thaliana5 Euglenagracilis5 C-ano+hora +arado9a5'listhodiscus luteus5 !ordeum 8ulgare5


35/86

Rhodo*acter ca+sulatus5 "-nechoc-stis.P.C.C. 0O35 Ana*aena 8aria*ilis5

'dontella sinensis5 &or+h-ra +ur+ureaand 0l-cine ma9 5ith GenBan>acceion n8m$er R21970, -073,61799, 60O, -3O21, 32100,-20, R1110,-31, %920, R0773, -3OO,

%O7 reectiel/. he three4+APae moti* are indicated (A, B andC) and the mot imortant reid8e areho5n in red. Q # @ &ndicate comletel/coneredreid8e and Q @ indicate coneratie8$tit8tion. Lo5ercae Q K @ indicateamino acid that are not conered. hiali+nment 5a created 8in+ ro+ramin the GCG ro+ram ac>a+e.ea, "-nechoc-stis and Rhodo*actere8ence reectiel/E,0,39F. 'ne interetin+ *eat8re o* all

the % 8$8nit i therolinerich re+ion at the centre,*ollo5ed $/ a hi+hl/ char+edtretch o* amino acid. Proline reid8eare tho8+ht to $eimortant in rotein±rotein interaction

EF and thi re+ion


36/86

ma/ $e imortant in interactin+ 5ith the: or & 8$8nit. ?i+8re3 ho5 a %otlot comarion E1F o*

the 8$8nit & conen8e8ence ho5n in ?i+8re 1 5ith a8$8nit%conen8 +enerated*rom the three % 8$8nit e8ence.%ia+onal line on thi?i+8re ho5 re+ion o* imilarit/

$et5een the t5o comarede8ence, and it i o$io8 that the Tterminal o* the% 8$8nitho5 imilarit/ to the & 8$8nit. he %8$8nit *rom ea and"-nechoc-stis (i.e. or+anim that ma>eChl) hae retained allthree 4+APae moti* (ee ?i+8re 2),5hile the Rhodo*acter %8$8nit aear to hae lot all eKcetthe econd moti*. he4+APae moti* on the % 8$8nit ma/$e er/ i+nicant in

that the/ +ie the rt indication thaten/me *rom Bchl andChl*ormin+ or+anim mi+ht hae8$tantial dierence. &n*act, a dic8ed reio8l/, hi+herlant hae a re8irement


37/86

*or AP in $oth the actiation andchelation te and, inceonl/ the actiation te ma/ 8tilie

APEF intead o* AP, thedata 8++et that thee APre8irement are earate. Poi$l/the %$o8nd AP i 8ed in one te and&$o8nd AP *or theother. Wh/ doe the $acterial en/me

not hae the econd AP$indin+ite I &* the % 8$8nit articiate inre+8lation, there ili>el/ to $e a dierence in it roertie$et5een Rhodo*acter and Chlrod8cin+ ecie 5hich *ace8$tantial dierence incontrollin+ haem and BchlChl /ntheiDoi$l/ the ariationin % e8ence reect thi. here are nine co8nterart o* the :8$8nit in GenBan> thatran+e in ie *rom 123 to 13 >%a. 'nl/

e o* thee are 4+chelatae8$8nit, 5ith the remainder encodin+Cochelatae8$8nit or 8tatie Tichelatae8$8nit (ee $elo5). hee8ence identitie o* the 4+chelatae

: 8$8nit ran+e *rom


38/86

$et5een 3O, comarin+ Rhodo*acter5ith $arle/, and 8 toO0, comarin+ Ara*ido+sis 5ith

Antirrhinum (nadra+on).?i+8re 1 ho5 the conen8 e8ence*or the 4+chelatae :8$8nit and the reid8e that are1conered in the e8enceo* all nine co8nterart o* the : 8$8nit.

A *8nction *or theerotein cannot $e 8rmied *rom acomarion 5ith othere8ence o* >no5n *8nction. :o5eer,the *8nction o* : itho8+ht to inole $oth the $indin+ o*Proto, $aed on $iochemicalo$eration E,21F and the $indin+ o*the 4+


39/86

/igure 3 .ot+lot of the .su*unitconsensus 6ith the Isu*unit consensus he aKi n8m$er are the amino acid

reid8e tartin+ *rom the Ttermin8. he ro+ram in the GCG ro+ramac>a+e 5ere 8ed to hel create thidia+ram.Co


40/86

and ea are the ame, the e8encedierence in the %8$8nit ma/ $e related to re+8lation

rather than mechanim. hi in*ormation can $e incororated intomodel o* ho5 4+chelataei oeratin+, and ha $een 8ed $elo5 inthe dic8iono* the mechanim.

A M'.EL /'R MgC!ELATA"E ACTIVIT)Com$inin+ in*ormation *rom $iochemicalaa/, chemicalmodel and +ene e8ence comariono* an en/me can +ieome ini+ht into ho5 that en/me i5or>in+. While thi i tr8e*or 4+chelatae, reearch on thien/me i at an earl/ ta+e andthe +a in o8r >no5led+e o* thien/me are conidera$leD an/model rooed 8nder theecirc8mtance ho8ld there*ore $e

conidered a hi+hl/ ec8latie. here*ore, the model that 5ereent in cheme 3 i roiional, $8t iconitent 5ith the datathat are aaila$le in the c8rrentliterat8re.


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he rt te in the reaction iactiation. &n thi te, 5e8++et that & i a dimer 5hich interact

5ith a % 8$8nit. ince% aear to $ehae a an a++re+ateE19,2OF, thi te i ho5na inolin+ the diociation o* a in+le %8$8nit *rom thea++re+ate; a roce 5hich ma/ $e

reciitated $/ the reenceo* & dimer and AP. hi te re8ireAP or APEF andco8ld inole rotein hohor/lation.:o5eer, it ho8ld $enoted that there i no direct eidence *orrotein hohor/lationin the actiation te at thi timeD thearticiation o* a rotein>inae ha $een incl8ded olel/ in anattemt to incororate theAPEF data into the model. ince $oth %and & hae AP$indin+

ite (at leat in Chlcontainin+or+anim), either 8$8nitco8ld $e actin+ a a rotein >inaeD inthe cheme, *P denote ahohor/lated &E% comleK and i notintended to 8++et


42/86

5hich o* the t5o 8$8nit ihohor/lated. he rod8ct o* thert te i an actiated &no5n.

he econd te o* catal/ii 4+


43/86

catal/i, and the % 8$8nit onl/ inactiationD thi i $aedolel/ on the tiochiometr/ o* the

8$8nit E0,21F rather thanroertie o* the 8$8nit. &* thi modeli correct, one 5o8ldeKect a lar+e comleK to *orm in thereence o* Proto, AP and4+


44/86


45/86


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maintain the reorted ratio o* 1:; 1&2E21F.en/micall/. :o5eer, the t5o clae o*

?e(&&)containin+tetra/rrole, 5hich are the haem andirohaem, are *ormeden/micall/ 5ith earate*errochelatae catal/in+ the inertiono* the ?eo* an ener+eticre8irement *or ?e


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rin+ contraction E3F. &n&ro+ion*acterium shermanii, aCochelatae

act $e*ore the contracted corrin rin+ i*ormed, andthe en/me aear to $e a in+leol/etide that doe notre8ire AP EF. &n contrat, in&seudomonas denitriGcans the

metal ion i inerted a*ter thecontraction o* the corrin rin+,5hich, d8e to the maller ie o* therin+, i a le *ao8ra$lereaction in ener+etic termD thi Cochelatae i APre8irin+and conit o* t5o earate rotein,Co$T and Co$, made8 *rom three dierent 8$8nit E2F. he Co$T o* the co$altochelatae,5hich i a co8nterart o* Bch: E0,OF,$ind $oth thetetra/rrole and the Co


48/86

the remainder o* the rotein ho5 no*8rther e8ence imilarit/to &. he third Co$ 8$8nit ha no

e8ence imilarit/ to the %8$8nit o* 4+chelatae. At the en/meleel, the @ m al8e *orthe Cochelatae 5ere O n4 *orh/dro+eno$/rinic acid a,cdiamide, 22 l4 *or AP E2F 5hich are

comara$le 5ith @ mal8e o* 4+chelatae o* 30 n4 or 13n4 *or Proto, and100 l4 or 3 l4 *or AP ($acterial andlant reectiel/)E21,2OF. he @ m al8e *or the 4+


49/86

co*actor. :o5eer, $aed on thetetra/rrole eKcreted $/m8tant, it i 8rmied that Ti


50/86

tetra/rrole reorted in methano+enic$acteria, it i reaona$leto a8me that the co8nterart o* 4+

chelatae are in *act8$8nit o* the Tichelatae inoled inthe $io/nthei o* the?3 co*actor. A8min+ eol8tionar/arimon/, the Bch& andBch% co8nterart ma/ alo $e inoled

in the inertion o* Co


51/86

imilaritie, indicatin+ that the/ hare acommon ancetr/.L'CALI(ATI'% '/ MgC!ELATA"E

WIT!I% T!E C!L'R'&LA"T here i a de$ate a to 5hether theen/me *or Chl and haem/nthei are atiall/ earated *romeach other 5ithin thechlorolat, $/ comartmentation o* one

art o* the Chl ath5a/to the th/la>oid and another art to theeneloe mem$raneE7±1F. 8ch a diiion co8ldotentiall/ $e 8e*8l in re+8latin+the relatie amo8nt o* haem and Chl/ntheied in thechlorolat. hi oint 5a initiall/ raied$/ ?8eler et al., 5hocomared the inhi$ition attern o* 4+chelatae and the 4+Proto monometh/l eter c/clae(c/clae) in intact chlorolat

E7F. hi 5or> ho5ed that c/clae 5amore rotected *rom thenonermeant thiol+ro8 inhi$itor +chloromerc8ri$enene8lhonate(PC4B) than 4+chelatae. 'neoi$le interretation


52/86


53/86

c/clae lie 5ithin the chlorolat innereneloe, $8t that thec/clae i in a more h/droho$ic

enironment and i there*orele accei$le to PC4B.&t ha $een con*8in+ that the in .itroea chlorolat 4+chelataeaa/ 5a rened to incl8de a cr8deearation o*

th/la>oid *rom eneloeD thechlorolat 5ere l/ed in $8er5itho8t 4+oidremoed $/ a lo5eed inE31F. he remainin+ cr8de eneloe andtromal rotein 5erehi+hl/ actie, indicatin+ that 4+chelatae 5a aociated 5iththe eneloe mem$rane. To5, 5ith thein*ormation that 4+chelataei a ol8$le en/me in the a$ence o* 4+E,19,27F, theedata can $e reinterreted ; d8rin+ l/i

and remoal o* theth/la>oid, 4+chelatae 5o8ld hae$een ol8$le; on the8$e8ent addition o* 4+


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In .i.o, there i 8Mcient 4+oid earationtechni8e re8ire lo5 4+concentration to o$tain mem$rane5ith little crocontaminationE2,3F, it i not oi$le to localie 4+

chelatae 5ithinthe chlorolat $/ thee method. hi8etion ma/ $e ettled$/ in .itro imort t8die andimm8no+old la$ellin+. Alread/ theimort o* the : and & 8$8nit into thetroma o* Ara*ido+sischlorolat ha $een reorted, altho8+htheir denitie aociation5ith either the eneloe and th/la>oidha not /et$een ho5n EF.&n 8mmar/, 5hile there i no eidence

to ho5 that 4+chelataeaociate 5ith either the th/la>oid orthe eneloe,the en/me i certainl/ tranortedthro8+h the eneloe andinto the troma. he li>elihood that it

nal detination i the


55/86

th/la>oid i increaed $/ the recentreai+nment o* anotherChl$io/nthetic en/me, Pchlide

red8ctae, to the th/la>oidE,F. &nteretin+l/, Pchlide red8ctaehad reio8l/ $eenreorted to $e on the chlorolateneloe EOF, $8t thi ha no5$een eKlained a an imort

intermediate E0F, and the naldetination o* thi en/me i tho8+ht to$e the th/la>oid E,F.'nl/ t5o other orh/rin$io/ntheticen/me hae $eenreorted to $e on the eneloe;rotoorh/rino+en oKidae(5hich *8nction in $oth haem and Chl/nthei) E9F and*errochelatae E7F. ince theeen/me are alo *o8nd in lar+eamo8nt on the th/la>oid E9,F, it$e+ the 8etion o*

5hether their reence on the eneloemi+ht alo $e eKlaineda imort intermediate. h8 an/earate orh/rin$io/ntheticcaacit/ o* the eneloe i not certainat thi time.


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RE0LATI'% '/ MgC!ELATA"E A%./ERR'C!ELATA"E AT T!E$RA%C!&'I%T

he oition o* 4+chelatae at the$ranchoint o* haem andChl /nthei, a 5ell a it tat8 a theen/me *or the rtcommitted te o* Chl /nthei, ma>e4+chelatae a

rime candidate *or ti+ht re+8lation.&nitial idea o* ho5 4+chelataeand the Chl $ranch 5ere re+8latedcentred on4+chelatae $ein+ re+8lated $/*eed$ac> inhi$ition $/ one o* the intermediate on the 4+ $ranch o*the ath5a/ E11F. hi i8orted $/ the recent o$eration thatthe articial in .i.oeleation o* 4+Proto and 4+Protomonometh/l eter $/*eedin+ ALA to ea eedlin+ ca8ed a

9 decreae in the in.itromea8ra$le 4+chelatae actiit/ iniolated ea chlorolatEOF. :o5eer, 5hen theeintermediate 5ere directl/teted in chlorolat, and more recentl/

in a ol8$ilied 4+chelatae


57/86

rearation, no eidence *or *eed$ac>inhi$ition $/4+Proto, 4+Proto monometh/l eter or

Pchlide co8ld $e*o8nd E10,2OF. ince thee intermediatedo not directl/ inhi$it4+chelatae, the onl/ eKlanation *ortheir aarent inhi$itionin the ALA*eedin+ eKeriment i $/

red8cin+ the amo8nt o* one or more o* the 4+chelatae8$8nit reent. hi co8ld $eaccomlihed $/ either red8cin+ thetrancrit leel o* the 4+chelatae+ene or aderel/ aectin+ the imorto* the 8$8nitinto the chlorolat andor increain+the t8rnoer o* one ormore o* the 4+chelatae 8$8nit. he adent o* the in .itro aa/ anddetermination o* the t5otenat8re o* the reaction reent another

otion *or thecontrol o* the 4+chelatae. Co8ld theinitial actiation, 5hichcan $e catal/ed $/ APEF, inole arotein >inae E2F I With8re, eKreed recom$inant rotein

thi idea i no5 readil/


58/86

teta$le $/ the inc8$ation o* & and %8$8nit 5ith radiola$elledAPEF.

Altho8+h no mechanim *or the directre+8lation o* 4+chelataeha $een reorted, the oerall re+8lationo* 4+chelatae and *errochelatae ha$een addreed. &n theor/, i*

*errochelatae and 4+chelataecomete *or the ame ool o* 33< C. J. Wal>er and ". %. Willo58$trate, their relatie actiitieho8ld $e coordinated to*ao8r one or the other ath5a/accordin+ to the reailin+demand o* the chlorolat. here ha$een onl/ one recentt8d/ in 5hich the relatie actiitie o*thee t5o en/me 5ereim8ltaneo8l/ etimated in intactchlorolat, allo5in+ thi

aect o* re+8lation to $e eKaminedE9F. When haem and4+Proto /nthei 5ere comared in eachlorolat, it 5aclear that in the reence o* AP there5a a dratic red8ction in


59/86

haem /nthei and 4+Proto /nthei5a *ao8red oer haem/nthei $/ a *actor o* a$o8t . &n

addition, $eide allo5in+4+%a ($aed on +elltration) E0F. hi 5or> ha

no5 $een *ollo5ed 8 5ith t8die onrecom$inant *errochelataecloned *rom c8c8m$er (. 4a8da,eronal comm8nication).A+ain, thi en/me 5a directl/ inhi$ited$/ AP, 5ith 1 m4


60/86

AP inhi$itin+ $/ . he eKreed*8ion rotein 5aol8$le, and imm8nolocaliation t8die

on c8c8m$er and inach("+inacia oleracea) chlorolat8++eted that it 5alocalied to the chlorolat troma in .i.o.AP inhi$ition o* *errochelatae 5a cometitie 5ith

reect to $oth orh/rinand ?e


61/86

either haem or haem rec8ror tomitochondria and the ret o* the cell. he eX8K o* haem and other

orh/rin *rom iolatedchlorolat ha $een o$eredE01±03F . Poi$l/ there are t5ohaem/nthei ath5a/ in thechlorolat; one *or hoto/nthetic8e and the other *or non

hoto/nthetic andoreKtrachlorolatidic haem. Perha themore ol8$le *orm o* theen/me i aociated 5ith thehoto/nthetic ath5a/. Whilehi+hl/ ec8latie, it $e+ the 8etiono* 5hether the mem$rane$o8nd*errochelatae i alo inhi$ited $/ AP.&* *errochelatae and 4+chelataecomete *or the ame oolo* Proto, another *actor 5hich m8t $eta>en into acco8nt itheir relatie >inetic contant.

?errochelatae in ea ha a @ m*or Proto o* 2. l4 and a V maK o* a$o8t.2 nmolEminYVEm+ o* ChlYV EF. 4+chelatae in ea haa$o8t the ame V maK a*errochelatae E9F, and initiall/ the @ m

*or Proto 5a tho8+ht to


62/86

$e a$o8t the ame a that *or*errochelatae E9F, $8t morerecentl/ aa/ condition 5ere *8rther

otimied 5ith ol8$iliedrearation to o$tain a $etter etimateo* the >inetic contantand the @ m *or Proto 5a reetimateda a$o8t 13 n4 E2OF. o $rin+ thee data to+ether, it eem

that 5e can $e condento* t5o mechanim 5hich control the 8Ko* intermediatethro8+h the $ranchoint. ?irt, thereence o* AP, 5hich ia$ol8tel/ re8ired *or 4+chelataeactiit/, alo inhi$it *errochelatae.econdl/, the @ m o* 4+chelatae *orProto i lo5eno8+h that it ho8ld o8tcomete*errochelatae *or theircommon 8$trate. hi ict8re o* there+8lation o* the $ranchoint

i *ar *rom comlete, ince the oi$ilit/that 4+chelataei re+8lated $/ rotein hohor/lationma/ /et hae to $e ta>eninto acco8nt. &n addition, there ieidence to 8++et that the %


63/86

8$8nit o* the en/me aociate 5ithri$oome in $oth hi+herlant and Rhodo*acter E19F. hi re8lt

5a not conrmed $/Gorchein, 5ho reorted that l/edRhodo*acter cell had actiit/in the hi+heed 8ernatant E0F Dho5eer, the l/i $8erincl8ded AP, 5hich co8ld otentiall/

aect the *ractionation o* the % comonent. While the aociationo* % 5ith the ri$oomeco8ld $e *ort8ito8, the en/me 5a aloinhi$ited $/ chloramhenicol,raiin+ the interetin+ oi$ilit/ thatthere ma/ $eome connection $et5een the re+8lationo* rotein /nthei ando* 4+chelatae. Another leel o* controlma/ $e +ene trancrition,and $e*ore an oerall model *orre+8lation i dic8ed,

c8rrent 5or> on the re+8lation o*trancrition 5ill $e dic8ed.RE0LATI'% '/ $ACTERIAL 0E%ETRA%"CRI&TI'% he +ene *or 4+chelatae in R.ca+sulatus, R. s+haeroides and


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Chloro*ium .i*rioforme are cl8tered5ith other Bchl$io/nthetic+ene and, *or Rhodo*acter at leat,

5ith the hoto/tem$io/nthetic +ene E3,0,00F. &nRhodo*acter , the *chIand *ch. +ene are art o* the ameoeron, 5herea the *ch!i trancri$ed earatel/. &n Chloro*ium

.i*rioforme the *ch!,*chI and *ch. are on the ame oeronE3F. Rhodo*acter can +ro5either hoto/ntheticall/ or aero$icall/8in+ a *ermenta$lecar$on o8rce, in 5hich cae thehoto/tem and i+ment arenot /ntheied. he re+8lation o* the+ene in the Rhodo*acter hoto/nthetic +ene cl8ter ha $eenreie5ed recentl/ E07,0OFand 5ill onl/ $e dic8ed $rie/. -nderanaero$ic condition,

eKreion o* thee i+ment$io/nthetic+ene i co8led 5ithother +ene inoled in hoto/ntheia art o* oerlain+trancritional 8nit termed8eroeron. he re+8lation o* the


65/86

hoto/tem +ene i $/ an anaero$icind8ction circ8it, 5hereathe re+8lation o* the i+ment

$io/nthetic +ene i $/ an aero$icrereion circ8it inolin+ a %TA$indin+ redoKenin+ roteinnamed CrtJ.At the rotein leel not m8ch i >no5na$o8t the re+8lation o*

4+chelatae in hoto/nthetic $acteria.'ne oi$le controlmechanim ha $een 8++eted $/ thendin+ that, 5hen the :8$8nit *rom Rhodo*acter ca+sulatus ieKoed to li+ht andoK/+en, the $o8nd Proto i conertedinto hotorotoorh/rin. hi orh/rin *orm a er/ ti+ht(oi$l/ coalent) attachmentto the:8$8nit and render it inactie Dthi ma/ $e an additionalmechanim *or the inhi$ition o* Bchl

$io/nthei $/ hi+h oK/+entenion and li+ht E09F. Additionall/, it ha$een o$ered that, in.itro, the reence o* the : 8$8nitincreae the actiit/ o* theneKt en/me in the ath5a/, namel/ the

meth/ltran*erae E7F.


66/86

&nteretin+l/, in 5hole Rhodo*acter cell,it 5a *o8nd that 4+chelataeactiit/ 5a onl/ mea8ra$le i* "

adeno/lmethionine5a incl8ded to allo5 themeth/ltran*erae to oerate E12F. hee data 8++et that there ma/ $e acloe lin>a+e $et5eenthee en/me in .i.o and raie the

oi$l/ that the : 8$8niti inoled in re+8latin+ another en/mein the ath5a/.!I0!ER&LA%T 0E%E RE0LATI'%'nl/ the chl! and I +ene hae $eenaaila$le *or trancritionalt8die to date, and o in*ormation onthe re+8lation o* the8$8nit at thi leel i not comlete.:o5eer, trancritionalre+8lation o* the & and : 8$8nit ha$een loo>ed at in $oth+reenin+ ti8e and a a *8nction o*

di8rnal ariation. he eKreion o* the chl! and chlI+ene in deeloin+ lanti ind8ced $/ li+ht. When dar>adated Ara*ido+sis and o/$ean(0l-cine ma9 ) 5ere tran*erred to li+ht

*or 2 h, the chlI trancrit


67/86

5a ind8ced 8 to 0*old comared 5iththe li+htadatedlant E9,1F. When etiolated $arle/

eedlin+ 5ere tran*erred toli+ht, the chl! trancrit increaed 2*old a*ter h and remained4echanim and re+8lation o* 4+chelatae 331

Proto ALA*l(tamateMgProto aemMP+PchlideChlorophyll,aProto ALA*l(tamateMgProto aem

MP+PchlideChlorophyll,bProto ALA*l(tamate


68/86

MgProto aemMP+Pchlide

Chlorophyll,c"cheme Model for the regulation of Chland haem *ios-nthesis in the chloro+last he $ar at the to o* the chemerereent the time o* da/ ran+in+ *rom

dar> to li+ht condition. he 5idth o*the arro5 +oin+ do5n rereent therelatie 8K o* intermediate, 5hile theredarro5 ointin+ to a oition in theath5a/ ho5 the leel o* rereion atthat oint in the ath5a/. he red arro5in the in> $oK containin+ : indicatethe amo8nt o* *8nctional : 8$8nitenterin+ the chlorolat. %otted lineindicate a ne+li+i$le 8K or eect.A$$reiation; 4P!, 4+rotoorh/rinmonometh/l eter. (a) ho5 the

it8ation N8t a*ter the tart o* the dar>eriod.(*) ho5 the it8ation N8t a*ter thetart o* the li+ht eriod, and (c) ho5the it8ation at the end o* the li+hteriod, a decri$ed in the teKt.

at thi leel D in thi /tem, theind8ction o* the trancrition o*


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& 5a not o rono8nced, 5ith onl/ a 2*old increae a*ter O hand a ret8rn to the 8nind8ced leel a*ter

2 h E7F. hi correlated5ith a *old increae in mea8ra$le 4+chelatae actiit/ a*ter0 h in the li+ht. o date, the oneeKcetion to thi +eneral atterno* li+ht ind8ction i the eKreion o* chlI

+ene o* Euglena gracilis,5hich 5a reorted to $e contit8tieDho5eer, thi eKcetionma/ $e d8e to the chlI +ene $ein+chlorolatencoded in thior+anim E71F. he eKreion o* thee +ene in lant+ro5n in a li+htdar>c/cle contrat 5ith the li+htind8cedeKreion in etiolatedti8e. &n $oth $arle/ and Ara*ido+sis,the chl! trancrit leelhad a$o8t a *old di8rnal ocillation

5ith the trancrit leel$e+innin+ to rie aroK. h $e*oreda5n, reachin+ a maKim8m1±3 h into the li+ht hae, then *allin+a+ain E,7F. &n $arle/, adi8rnal 8ct8ation in 4+chelatae

actiit/ 5a mea8red 5ith a


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trilin+ o* actiit/ *rom reda5n tomaKimal actiit/ 1±3 h otda5nE7F D no actiit/ mea8rement 5ere

made in the Ara*ido+sist8d/ EF. &n Antirrhinum ma?us a imilarit8ation 5a o$ered,eKcet that trancrit leel decreaedto almot ero $/ the endo* the li+ht eriodD ho5eer, a in

Ara*ido+sis and $arle/,trancrit leel came 8 d8rin+ thedar> hae EOF. &n $arle/, thi5or> 5a eKtended, and contin8ed8ct8ation in trancritleel 5ere o$ered i* the lant 5eretran*erred to contin8o8li+ht, indicatin+ that the trancrition5a 8nder the control o* a circadian cloc>. he $ehaio8r o* the &trancrit 8nder di8rnalcondition 5a 8ite dierent *rom thato* the : trancrit ; in

$oth $arle/ and Ara*ido+sis there 5alittle or no ocillationE,7F.With the a8mtion that the hi+herlant decri$ed a$oeare re+8latin+ 4+chelatae in imilar

5a/, t5o theme emer+e


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*rom thi 5or>. ?irtl/, the : 8$8niteem to 8ct8ate morethan the & 8$8nit 8nder +reenin+

condition or d8rin+ di8rnal+ro5th. econdl/, the & 8$8nit iind8ced $/ li+ht i* the ti8e ietiolated, $8t once the chlorolat haedeeloed, there i er/little eect o* li+ht on thi 8$8nit@

trancrition. While it i notoi$le to t % into thi attern a /et,it i reaona$le to a8methat the re+8lation o* thi en/me i inart achieed $/ re+8lationo* the amo8nt o* the : 8$8nit in thechlorolat.M'.EL /'R T!E RE0LATI'% '/ MgC!ELATA"E I% !I0!ER&LA%T" he data a$oe can $e 8ed to 8++et amodel *or the oerallre+8lation o* the 4+chelatae ath5a/

in hi+her lant +ro5nin li+ht±dar> c/cle. cheme ho5the it8ation at the end o* 332 C. J. Wal>er and ". %. Willo5the li+ht hae, 5here the chl!trancrit leel i lo5 and $e+in


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to rie at the tart o* the dar> hae. hi re8lt in li+htl/ hi+her4+chelatae actiit/ and a li+ht hi*t in

the 8K o* theintermediate *rom the haem $ranch tothe 4+ $ranch. :o5eer,4+ containin+ intermediate $e+in toacc8m8late $eca8e theconerion o* Pchlide to Chlide re8ire

li+ht EF. A 4+orh/rin acc8m8late the/ decreaethe amo8nt o* the :8$8nit that can enter the chlorolat,reentin+ their o5n/nthei. &n cheme ($), at the tart o* the li+ht hae, theeremainin+ 4+ intermediate are 8tiliedto ma>e Chl no5 thatthe conerion o* Pchlide into Chlide canta>e lace. hireleae the $loc> on : 8$8nitacc8m8lation. he chl! trancrit

leel and leel o* : 8$8nit reach amaKim8m earl/ inthi hae. A : 8$8nit acc8m8late inthe chlorolat, the/ca8e the dierion o* nearl/ all theProto into the 4+ $ranchD


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and, ince haem t8rn oer in thechlorolat, thi 5ill alo ca8ea decreae in *ree haem leel. &n the

reence o* haem, ALA/nthei i inhi$ited E72F and o a thehaem leel *all in thihae, o 5ill the inhi$ition o* ALA/nthei $e remoed,re8ltin+ in a net increae in the 8K o*

intermediate into thetetra/rrole ath5a/. &n cheme (c),to5ard the end o* theli+ht hae, the chl! trancrit *allraidl/ to almot ero 5iththe re8ltant decreae in : 8$8nitleel and a $loc> in the o5o* intermediate into the 4+ $ranch o*the ath5a/. hi allo5Proto to $e dierted $ac> to haem/nthei a+ain, and theincreaed leel o* *ree haem inhi$it ALA/nthei and h8t

do5n the tetra/rrole ath5a/. he re+8lation decri$ed a$oe i ad/namic roce andcheme (a)±(c) onl/ rereentecic ta+e thro8+h the da/. he model i alo incomlete and 5ill

ro$a$l/ need to $e


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eKtended to incororate other *actor8ch a the oi$ilit/ o* the hohor/lation o* 4+chelatae

8$8nit and theAPA%Pratio o* the chlorolat.C'%CL"I'%" A%. &ER"&ECTIVE"&n the 199, 5or> on 4+chelatae hamade a h8+e lea *romthe rt in .itro aa/ to clonin+ and

eKreion o* the +eneinoled. he c8rrent tat8 o* o8r>no5led+e can $e 8mmaried$/ concl8din+ that there are threerotein inoled in 4+chelataeand that the reaction itel* ta>e lace int5o te.With the recent aaila$ilit/ o* 8riedrecom$inant rotein, 5eanticiate that eta$lihin+ themechanim o* 4+chelatae 5illhaen er/ oon. &t 5ill $e interetin+to ole nall/ the 8le

o* 5h/ AP i needed in the reaction,5hich ma/ $e oled $/ in.itro la$ellin+ t8die o* the 8$8nit.Alo, it ho8ld $e oi$leto determine the role o* each 8$8nit inthe catal/tic mechanim.


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We than> %r. J. %. Weintein, %r. . &.Beale, %r. J. Ch8c> and %r. . 4. 4e/er*or

their critical readin+ o* the man8crit$e*ore it 8$miion and *or man/hel*8lcomment. We alo than> the man/a8thor 5ho +ae 8 reie5 o* their5or> in

the re. C. J. W. ac>no5led+e 8ort*rom the -.. %eartment o* !ner+/(+rantno. %!?G9!"217), and ". %. W.ac>no5led+e 8ort *rom the Tationalcience ?o8ndation (+rant no. T?4CB9091) and the -. . %eartment o* !ner+/ (+rant no. %!?G2OO!"1391O).RE/ERE%CE"1 %aile/, :. A. (199) in Conerion o*Coroorh/rino+en to Protohaem in:i+her

!8>ar/ote and Bacteria ; erminal hree!n/me (%aile/, :. A., ed.), .123±102,4cGra5:ill, Te5 Sor>2 Wal>er, C. J. and Weintein, J. %. (1991)Proc. Tatl. Acad. ci. -..A. FF,

7O9±793


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3 Peteren, B., 4Zller, 4., t8mmann, B.and :ennin+en, . (1990) :eredita12;,

93±90 Gi$on, L. C. %., Willo5, ". %.,annan+ara, C. G., on Wetttein, %. and:8nter, C.T. (199) Proc. Tatl. Acad. ci. -..A. =2,191±19

Gi$on, L. C. %., 4arrion, J. L., Leech,". 4., Jenen, P. !., Baham, %. C.,Gi$on,4. and :8nter, C. T. (1990) PlantPh/iol. 111, 01±710 Jenen, P. !., Gi$on, L. !., :ennin+en,. W. and :8nter, C. T. (1990) J. Biol.Chem. 271, 10002±100077 Jenen, P. !., Willo5, ". %., Peteren,B. L., Uoth>necht, -. C., t8mmann, B.4.,annan+ara, C. G., on Wetttein, %. and:ennin+en, . W. (1990) 4ol. Gen.

Genet.2;


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9 onc, C., 4a/erho*er, "., oncalman, R., Ta5rath, C., "ei, B., "edei,G. P. and

chell, J. (199) !4B' J. =, 1337±1301 Ta>a/ama, 4., 4a8da, ., ato, T., Sama+ata, :., Bo5ler, C., 'hta, :., hioi, S.and a>ami/a, . (199) Biochem.Bioh/. "e. Comm8n. 21;, 22±2O

11 Beale, . &. and Weintein, J. %. (199)in etra/rrole 4eta$olim inPhoto/nthetic'r+anim (%aile/, :. A., ed.), .2O7±391, 4cGra5:ill, Te5 Sor>12 Gorchein, A. (1972) Biochem. J. 127,97±1013 Gorchein, A. (1973) Biochem J. 13,O33±O1 Catel*ranco, P. A., Weintein, J. %.,ch5arc, ., Pardo, A. %. andWeelman, B. !.(1979) Arch. Biochem. Bioh/. 1=2,

92±9O1 Pardo, A. %., Chere>in, B. 4.,Catel*ranco, P. A., ?rancechi, U. ". andWeelman,B. !. (19O) Plant Ph/iol. :;, 90±9010 Wal>er, C. J. and Weintein, J. %.

(1991) Plant Ph/iol. =;, 11O9±1190


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17 "ichter, 4. L. and "ienit, . G.(19O2) Biochim. Bioh/. Acta 717,2±20

1O Lee, :. J., Ball, 4. %., Parham, ". and"e$ei, C. A. (1992) Plant Ph/iol. ==,113±1119 annan+ara, C., Uoth>necht, -.,:anon, 4. and on Wetttein, %.(1997) 4ol. Gen.

Genet. 2;, O±922 Wal>er, C. J. and Weintein, J. %.(199) Biochem. J. 2==, 277±2O21 Willo5, ". %., Gi$on, L. C. %.,anan+ara, G. C., :8nter, C. T. and onWetttein,%. (1990) !8r. J. Biochem. 23;, 3O±322 Al$erti, 4., B8r>e, %. :. and :eart, J.!. (199) in AnoK/+enic hoto/nthetic$acteria, (Blan>enhi, ". !., 4adi+an,4. . and Ba8er, C. !., ed.), .1O3±110,l85er Academic P8$liher, %ordrecht

23 Bolliar, %. W., 88>i, J. S., Beatt/, J. ., %o$ro5ol>i, J. 4. and Ba8er, C. !.(199) J. 4ol. Biol. 237, 022±02 Bolliar, %., Jian+, R., Ba8er, C. andBeale, . (199) J. Bacteriol. 17:,

29±290


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(1971) in he Genetic Control o*Chlorolat %eeloment in Barle/(Boardmann, T.,Linnana, A. and millie, "., ed.), .2±223, Torth:olland, Amterdam20 :ennin+en, . W., Bo/nton, J. !. and

on Wetttein, %. (1993) Biol. >r. .%an.Uiden>. el>. 2, 1±3927 Wal>er, C. J. and Weintein, J. %.(199) Ph/iol. Plant. =, 19±22O G8o, "., L8o, 4. and Weintein, J. %.(1997) Plant Ph/. !lectron. A$tr. 7329 Jaco$, J. 4., Jaco$, J. J., Borot, . !.and G8erinot, 4. L. (199) Arch.Biochem.Bioh/. 2Fer, C. J., :8, L. ". andWeintein, J. %. (1992) Plant Ph/iol.Biochem. 3


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3 ?leicher, !., Choi, !., :am$ri+ht, P.and tone, A. (190) &nor+. Chem. 3,12O±12O730 ohler, ., %el5iche, C., %enn/, P., ilne/, L., We$ter, P., Wilon, "., Palmer, J. and"oo, %. (1997) cience 27;, 1O±1O937 oonin, !. U. (1993) T8cleic Acid "e.21, 21±273O oonin, !. U. (1997) cience 27;,1O9±194echanim and re+8lation o* 4+chelatae 333

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Mechanism and Regulation Mg

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