Qn1)

Vertebrate Sequence
Sequence 1
>gi|632808|gb|AAB31500.1| pyruvate carboxylase [Homo sapiens]
MLKFRTVHGGLRLLGIRRTSTAPAASPNVRRLEYKPIKKVMVANRGEIAIRVFRACTELGIRTVAIYSEQ
DTGQMHRQKADEAYLIGRGLAPVQAYLHIPDIIKVAKENNVDAVHPGYGFLSERADFAQACQDAGVRFIG
PSPEVVRKMGDKVEARAIAIAAGVPVVPGTDAPITSLHEAHEFSNTYGFPIIFKAAYGGGGRGMRVVHSY
EELEENYTRAYSEAWPAFGNGALFVEKFIEKPRHIEVQILGDQYGNILHLYERDCSIQRRHQKVVEIAPA
AHLDPQLRTRLTSDSVKLAKQVGYENAGTVEFLVDRHGKHYFIEVNSRLQVEHTVTEEITDVDLVHAQIH
VAEGRSLPDLGLRQENIRINGCAIQCRVTTEDPAPTFQPDTGRIEVFRSGEGMGIRLDNASAFQGAVISP
HYDSLLVKVIAHGKDHPTAATKMSRALAEFRVRGVKTNIAFLQNVLNNQQFLAGTVDTQFIDENPDVFQL
RPAQNRAQKLLHYLGHVMVNGPTTPIPVKASPSPTDPVVPAVPIGPPPAGFRDILLREGPEGFARAVRNH
PGLLLMDTTFRDAHQSLLATRVRTHDLKKIAPYVAHNFSKLFSMENWGGATFDVAMRFLYECPWRRLQEL
RELIPNIRFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDVFRVFDSLNYLPNMLLGMEAAGSAGGVV
EAAISYTGDVADPSRTKYSLQYYMGLAEALVRAGTHILCIKDMAGLLKPTACTMLVSSLRDRFPDLPLHI
HTHAPSGAGVAAMLACAQAGADVVDVAADSMSGMTSQPSMGALVACTRGTPLDTEVPMERVFDYSEYWEG
ARGLYAAFDCTATMKSGNSDVYENEIPGGQYTNLHFQAHSMGLGSKFKEVKKAYVEANQMLGDLIKVTPS
SKIVGDLAQFMVQNGLSRAEAEAQAEELSFPRSVVEFLQGYIGVPHGGFPEPFRSKVLKDLPRVEGRPGA
SLPPLDLQALEKELVDRHGEEVTPEDVLSAAMYPDVFAHFKDFTATFGPLDSLNTRLFLQGPKIAEEFEV
ELERGKTLHIKALAVSDLNRAGQRQVFFELNGQLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPGKV
IDIKVVAGAKVAKGQPLCVLSAMKMETVVTSPMEGTVRKVHVTKDMTLEGDDLILEIE

Sequence 2
>gi|45383466|ref|NP_989677.1| pyruvate carboxylase [Gallus gallus]
MSQLCVPRGGRALLGAWRLPLLRPPPGSVRSASCQPIRKVLVANRGEIAIRVFRACTELGLRTVAVYSEQ
DTGQMHRQKADEAYLVGRGLPPVQAYLHVPDIIRVARENAVDAIHPGYGFLSERADFAQACVDAGVRFVG
PPPEVVRKMGDKVEARSIAIAAGVPVVPGTSAPVATLGEAQDFAARVGFPIIFKAAHGGGGRGMRAVRGP
QELEESFSRASSEALAAFGDGALFVEKLMERPRHIEGQILGDKHGNVVHLYERDCSIQRRHQKVVEIAPA
ARLDPQLRAQLASDAVRIAQQVGYENAGTVEFLVDRDGKHYFIEVNSRLQVEHTVTEEITGVDLVQAQLL
VAAGRSLSELGLQQDSVRVNGCAIQCRVTTEDPARGFQPDTGRIEVFRSGEGMGIRLDGASAFQGALISP
HYDSLLVKVIAHGPDQPSAAAKMSRALGEFRIRGVKTNIPFLQNVLAHPQFLGGAVDTQFIDENPELFHL
RPSQNRAQKLLHYLGHVMVNGPSTPLPVKAKAAVVEPVPPPVPMGSPPEGLRAVLQREGPAGFARALRGH
RGLLLXDTTFRDAHQSLLATRVRTRDLARIAPFVAHSLSPLCSMETWGGATFDVAMRFLHECPWERLREL
RRLVPNIPFQMLLRGANAVGYTNYPDNVIYRFCEVAAANGMDIFRIFDALNYLPNLLLGVEAVGRAGAVV
EAALSYTGDVADPTRTKYSLDYYLGLAKELVAAGTHILCIKDMAGLLTPAAARLLVSSLRDRFPDVPIHV
HTHDTAGAAIATLLAAANADADVVDVAVDAMSGMTSQPSMGALVACARGTPLDTGIALERVFEYSEYWEG
ARGLYAAFDCTATMKSGNADVYENEIPGGQYTNLHFQAHAMGLGHKFKEVKKAYAEANKLLGDLIKVTPS
SKVVGDLAQFMVQNGLSREEAEARADELSFPLSVVEFLQGYIGTPPGGFPEPFRSKVLKDLPRVEGRPGA
SLPPLDFEALSQELGARDGTPPSPEDLLSAALYPKVYAEFRDFTSTFGPVSCLGTRLFLEGPTIAEEFEV
ELERGKTLHIKALALGDLNAAGQREAFFELNGQLRSILVRDTQALKEMHVHPKADRSAKGQVGAPMPGEV
VEVRVKEGEAVEKGAPLCVLSAMKMETVVTAPRGGTVSRLHVRPGMSLEGDDLIAEIE

Sequence 3
>gi|1040974|gb|AAC52668.1| pyruvate carboxylase [Rattus norvegicus]
MLKFQTVRGGLRLLGVRRSSTAPVASPNVRRLEYKPIKKVMVANRGEIAIRVFRACTELGIRTVAVYSEQ
DTGQMHRQKADEAYLIGRGLAPVQAYLHIPDIIKVAKENGVDAVHPGYGFLSERADFAQACQDAGVRFIG
PSPEVVRKMGDKVEARAIAIAAGVPVVPGTNSPINSLHEAHEFSNTYGFPIIFKAAYGGGGRGMRVVHSY
EELEENYTRAYSEALAAFGNGALFVEKFIEKPRHIEVQILGDQYGNILHLYERDCSIQRRHQKVVEIAPA
THLDPQLRSRLTSDSVKLAKQVGYENAGTVEFLVDKHGKHYFIEVNSRLQVEHTVTEEITDVDLVHAQIH
VSEGRSLPDLGLRQENIRINGCAIQCRVTTEDPARSFQPDTGRIEVFRSGEGMGIRLDNASAFQGAVISP

HYDSLLVKVIAHGKDHPTAATKMSRALAEFRVRGVKTNIPFLQNVLNNQQFLAGIVDTQFIDENPELFQL
RPAQNRAQKLLHYLGHVMVNGPTTPIPVKVSPSPVDPIVPVVPIGPPPAGFRDILLREGPEGFARAVRNH
QGLLLMDTTFRDAHQSLLATRVRTHDLKKIAPYVAHNFNNLFSIENWGGATFDVAMRFLYECPWRRLQEL
RELIPNIPFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDVFRIFDSLNYLPNMLLGMEAAGSAGGVV
EAAISYTGDVADPSRTKYSLEYYMGLAEELVRAGTHILCIKDMAGLLKPAACTMLVSSLRDRFPDLPLHI
HTHDTSGSGVAAMLACAQAGADVVDVAVDSMSGMTSQPSMGALVACTKGTPLDTEVPLERVFDYSEYWEG
ARGLYAAFDCTATMKSGNSDVYENEIPGGQYTNLHFQAHSMGLGSKFKEVKKAYVEANQMLGDLIKVTPS
SKIVGDLAQFMVQNGLSRAEAEAQAEELSFPRSVVEFLQGYIGIPHGGFPEPFRSKVLKDLPRIEGRPGA
SLPPLNLKELEKDLIDRHGEEVTPEDVLSAAMYPDVFAQFKDFTATFGPLDSLNTRLFLQGPKIAEEFEV
ELERGKTLHIKALAVSDLNRAGQRQVFFELNGQLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPGKV
IDVKVAAGAKVVKGQPLCVLSAMKMETVVTSPMEGTIRKVHVTKDMTLEGDDLILEIE

Bacterial Sequence
Sequence 4
>gi|504618726|ref|WP_014805828.1| pyruvate carboxylase [Mycobacterium chubuense]
MFSKVLVANRGEIAIRAFRAVYELGAATVAVYPYEDRNSLHRSKADESYQIGVEGHPVRAYLNVDHIVST
ALDCGADAIYPGYGFLSENPELATACAAAGITFVGPSADVLELTGNKARAIAAARAAGLPVLASSQPSAD
VHELVAAAQSMEFPLFVKAVAGGGGRGMRRVAAAADLQEAIEAASREAESAFGDATVFLEQAVVNPRHIE
VQILADGSGEVIHLFERDCSVQRRHQKVIELAPAPNLEPALRARICDDAVAFARQIGYSCAGTVEFLLDE
RGQHVFIEMNPRIQVEHTVTEEITDVDLVSAQLRIAAGETLADLGLAQDTLQIHGAAIQCRITTEDPANG
FRPGTGRITGYRSPGGAGIRLDGGTNIGAEVTAHFDSMLVKLSCRGRDFDTAVRRARRAVAEFRIRGVST
NIPFLQAVLDDPDFQSGRVTTSFIDERPGLLTARSSADRGTKILNYLADVTVNQPHGPRPSAVYPRDKLP
VIDLTAAPPPGSKQRLTKLGPEGFALWMRESKTVGVTDTTFRDAHQSLLATRVRTSGLMRVAPYIARMTP
ELLSVECWGGATYDVALRFLKQDPWDRLAALREALPNICLQMLLRGRNTVGYTPYPEQVTSAFVEEAAQT
GVDIFRIFDSLNNIVAMRPAIDAVLNTGTTIAEVAMSYTGDLSDPGEDLYTLDYYLRLAEAIVDAGAHVL
AIKDMAGLLRAPAAATLVAALKSRFDLPVHVHTHDTPGGQLATYAAAWAAGADAVDGAAAPLSGTTSQPS
LSSIVAAAARTEFDTGLSLSAVCDLEPYWEALRKVYAPFESGLAAPTGRVYTHEIPGGQLSNLRQQAIAL
GLGDRFEDVENAYAGADRVLGRLVKVTPSSKVVGDLALALVGAGASAEEFAAEPGLYDLPDSVIGFLRGE
LGDPPGGWPEPLRTKALDGRGPAKPEQELTVEQEAVLAAPGPKRRAMLNHLLFAGPTAEFEAHREEFGDT
SRLSANQFFYGLRHGEEHRVTLEPGVELLIGLEAISDADERGMRTVMCIINGQLRPVMVRDRSIASDIPV
SERADKTNPDHVAAPFAGVVTVTVAQGDAVEAGQTLATIEAMKMEAAITATKAGTLNRIAVAATAQVESG
DLLMVLT

Fungal Sequence
Sequence 5
>gi|12044690|emb|CAC19838.1| pyruvate carboxylase [Aspergillus niger]
MAAPRQPEEAVDDTEFIDDHHDQHRDSVHTRLRANSAIMQFQKILVANRGEIPIRIFRTAHELSLQTVAV
YSHEDHLSMHRQKADEAYMIGKRGQYTPVGAYLAIDEIVKIALEHGVHLIHPGYGFLSENAEFARKVEQS
GMVFVGPTPQTIESLGDKVSARQLAIRCDVPVVPGTPGPVERYEEVKAFTDTYGFPIIIKAAFGGGGRGM
RVVRDQAELRDSFERATSEARSAFGNGTVFVERFLDRPKHIEVQLLGDNHGNVVHLFERDCSVQRRHQKV
VEIAPAKDLPADVRDRILADAVKLAKSVNYRNAGTAEFLVDQQNRYYFIEINPRIQVEHTITEEITGIDI
VAAQIQIAAGATLEQLGLTQDRISTRGFAIQCRITTEDPSKGFSPDTGKIEVYRSAGGNGVRLDGGNGFA
GAIITPHYDSMLVKCTCRGSTYEIARRKVVRALVEFRIRGVKTNIPFLTSLLSHPVFVDGTCWTTFIDDT
PELFALVGSQNRAQKLLAYLGDVAVNGSSIKGQIGEPKLKGDIIKPVLHDAAGKPLDVSVPATKGWKQIL
DSEGPEAFARAVRANKGCLIMDTTWRDAHQSLLATRVRTIDLLNIAHETSHALANAYSLECWGGATFDVA
MRFLYEDPWDRLRKLRKAVPNIPFQMLLRGANGVAYSSLPDNAIYHFCKQAKKCGVDIFRVFDALNDVDQ
LEVGIKAVHAAEGVVEATICYSGDMLNPSKKYNLPYYLDLVDKVVQFKPHVLGIKDMAGVLKPQAARLLI
GSIRERYPDLPIHVHTHDSAGTGVASMIACAQAGADAVDAATDSLSGMTSQPSIGAILASLEGTEHDPGL
NSAQVRALDTYWAQLRLLYSPFEAGLTGPDPEVYEHEIPGGQLTNLIFQASQLGLGQQWAETKKAYESAN

DLLGDVVKVTPTSKVVGDLAQFMVSNKLTAEDVIARAGELDFPGSVLEFLEGLMGQPYGGFPEPLRSRAL
RDRRKLDKRPGLYLEPLDLAKIKSQIRENYGAATEYDVASYAMYPKVFEDYKKFVAKFGDLSVLPTRYFL
AKPEIGEEFHVELEKGKVLILKLLAIGPLSEQTGQREVFYEVNGEVRQVSVDDKKASVENTARPKAELGD
SSQVGAPMSGVVVEIRVHDGLEVKKGDPIAVLSAMKMEMVISAPHSGKVSSLLVKEGDSVDGQDLVCKIV
KA
CLUSTAL 2.1 multiple sequence alignment
gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

MLKFRTVH---GGLRLLGIRRTSTAPAASPNVRR-LEYKPIKKVMVANRG
MSQLCVPR---GGRALLGAWRLPLLRPPPGSVRS-ASCQPIRKVLVANRG
MLKFQTVR---GGLRLLGVRRSSTAPVASPNVRR-LEYKPIKKVMVANRG
MAAPRQPEEAVDDTEFIDDHHDQHRDSVHTRLRANSAIMQFQKILVANRG
---------------------------------------MFSKVLVANRG
---------------------------------------MLDKIVIANRG
: *:::****

46
46
46
50
11
11

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

EIAIRVFRACTELGIRTVAIYSEQDTGQMHRQKADEAYLIG-RG-LAPVQ
EIAIRVFRACTELGLRTVAVYSEQDTGQMHRQKADEAYLVG-RG-LPPVQ
EIAIRVFRACTELGIRTVAVYSEQDTGQMHRQKADEAYLIG-RG-LAPVQ
EIPIRIFRTAHELSLQTVAVYSHEDHLSMHRQKADEAYMIGKRGQYTPVG
EIAIRAFRAVYELGAATVAVYPYEDRNSLHRSKADESYQIGVEG--HPVR
EIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVCIGPAP---SVK
**.:* :*: **. ***::. *
*
***: :*
.*

94
94
94
100
59
58

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

AYLHIPDIIKVAKENNVDAVHPGYGFLSERADFAQACQDAGVRFIGPSPE
AYLHVPDIIRVARENAVDAIHPGYGFLSERADFAQACVDAGVRFVGPPPE
AYLHIPDIIKVAKENGVDAVHPGYGFLSERADFAQACQDAGVRFIGPSPE
AYLAIDEIVKIALEHGVHLIHPGYGFLSENAEFARKVEQSGMVFVGPTPQ
AYLNVDHIVSTALDCGADAIYPGYGFLSENPELATACAAAGITFVGPSAD
SYLNIPAIISAAEITGAVAIHPGYGFLSENANFAEQVERSGFIFIGPKAE
:** : *: *
. ::********..::*
:*. *:** .:

144
144
144
150
109
108

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

VVRKMGDKVEARAIAIAAGVPVVPGTDAPITSLHEAH-EFSNTYGFPIIF
VVRKMGDKVEARSIAIAAGVPVVPGTSAPVATLGEAQ-DFAARVGFPIIF
VVRKMGDKVEARAIAIAAGVPVVPGTNSPINSLHEAH-EFSNTYGFPIIF
TIESLGDKVSARQLAIRCDVPVVPGTPGPVERYEEVK-AFTDTYGFPIII
VLELTGNKARAIAAARAAGLPVLASS-QPSADVHELV-AAAQSMEFPLFV
TIRLMGDKVSAIAAMKKAGVPCVPGSDGPLGDDMDKNRAIAKRIGYPVII
.:. *:*. *
..:* :..: *
:
:
:*::.

193
193
193
199
157
158

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

KAAYGGGGRGMRVVHSYEELEENYTRAYSEAWPAFGNGALFVEKFIEKPR
KAAHGGGGRGMRAVRGPQELEESFSRASSEALAAFGDGALFVEKLMERPR
KAAYGGGGRGMRVVHSYEELEENYTRAYSEALAAFGNGALFVEKFIEKPR
KAAFGGGGRGMRVVRDQAELRDSFERATSEARSAFGNGTVFVERFLDRPK
KAVAGGGGRGMRRVAAAADLQEAIEAASREAESAFGDATVFLEQAVVNPR
KASGGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYMEKYLENPR
** ******** *
:* :
: ** .**.: :::*: : .*:

243
243
243
249
207
208

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

HIEVQILGDQYGNILHLYERDCSIQRRHQKVVEIAPAAHLDPQLRTRLTS
HIEGQILGDKHGNVVHLYERDCSIQRRHQKVVEIAPAARLDPQLRAQLAS
HIEVQILGDQYGNILHLYERDCSIQRRHQKVVEIAPATHLDPQLRSRLTS
HIEVQLLGDNHGNVVHLFERDCSVQRRHQKVVEIAPAKDLPADVRDRILA
HIEVQILADGSGEVIHLFERDCSVQRRHQKVIELAPAPNLEPALRARICD
HVEIQVLADGQGNAIYLAERDCSMQRRHQKVVEEAPAPGITPELRRYIGE
*:* *:*.* *: ::* *****:*******:* *** : . :* :

293
293
293
299
257
258

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

DSVKLAKQVGYENAGTVEFLVDRHGKHYFIEVNSRLQVEHTVTEEITDVD
DAVRIAQQVGYENAGTVEFLVDRDGKHYFIEVNSRLQVEHTVTEEITGVD
DSVKLAKQVGYENAGTVEFLVDKHGKHYFIEVNSRLQVEHTVTEEITDVD
DAVKLAKSVNYRNAGTAEFLVDQQNRYYFIEINPRIQVEHTITEEITGID
DAVAFARQIGYSCAGTVEFLLDERGQHVFIEMNPRIQVEHTVTEEITDVD
RCAKACVDIGYRGAGTFEFLFE-NGEFYFIEMNTRIQVEHPVTEMITGVD
.. . .:.* *** ***.: ... ***:*.*:****.:** **.:*

343
343
343
349
307
307

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.

LVHAQIHVAEGRSLPDLGLRQENIRINGCAIQCRVTTEDPAPTFQPDTGR
LVQAQLLVAAGRSLSELGLQQDSVRVNGCAIQCRVTTEDPARGFQPDTGR
LVHAQIHVSEGRSLPDLGLRQENIRINGCAIQCRVTTEDPARSFQPDTGR
IVAAQIQIAAGATLEQLGLTQDRISTRGFAIQCRITTEDPSKGFSPDTGK
LVSAQLRIAAGETLADLGLAQDTLQIHGAAIQCRITTEDPANGFRPGTGR

393
393
393
399
357

gi|145175|gb|AAA23409.1|

LIKEQLRIAAG---QPLSIKQEEVHVRGHAVECRINAEDP-NTFLPSPGK 353
:: *: :: *
*.: *: : .* *::**:.:***
* *..*:

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

IEVFRSGEGMGIRLDNASAFQGAVISPHYDSLLVKVIAHGKDHPTAATKM
IEVFRSGEGMGIRLDGASAFQGALISPHYDSLLVKVIAHGPDQPSAAAKM
IEVFRSGEGMGIRLDNASAFQGAVISPHYDSLLVKVIAHGKDHPTAATKM
IEVYRSAGGNGVRLDGGNGFAGAIITPHYDSMLVKCTCRGSTYEIARRKV
ITGYRSPGGAGIRLDGGTNIG-AEVTAHFDSMLVKLSCRGRDFDTAVRRA
ITRFHAPGGFGVRWES-HIYAGYTVPPYYDSMIGKLICYGENRDVAIARM
* ::: * *:* :.
:..::**:: * . *
* :

443
443
443
449
406
402

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

SRALAEFRVRGVKTNIAFLQNVLNNQQFLAGTVDTQFIDENPDVFQLRPA
SRALGEFRIRGVKTNIPFLQNVLAHPQFLGGAVDTQFIDENPELFHLRPS
SRALAEFRVRGVKTNIPFLQNVLNNQQFLAGIVDTQFIDENPELFQLRPA
VRALVEFRIRGVKTNIPFLTSLLSHPVFVDGTCWTTFIDDTPELFALVGS
RRAVAEFRIRGVSTNIPFLQAVLDDPDFQSGRVTTSFIDERPGLLTARSS
KNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNIHYLEKKLGLQEK--.*: *: : *:.**: :
:: . * *
:::.
:

493
493
493
499
456
449

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

QNRAQKLLHYLGHVMVNGPTTP---IPVKASPSPTDPVVPAVP------I
QNRAQKLLHYLGHVMVNGPSTP---LPVKAKAAVVEPVPPPVP------M
QNRAQKLLHYLGHVMVNGPTTP---IPVKVSPSPVDPIVPVVP------I
QNRAQKLLAYLGDVAVNGSSIKGQIGEPKLKGDIIKPVLHDAAGKPLDVS
ADRGTKILNYLADVTVNQPHGP---RPSAVYPRDKLPVIDLTA--------------------------------------------------------

534
534
534
549
496

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

GPPPAGFRDILLREGPEGFARAVRNHPGLLLMDTTFRDAHQSLLATRVRT
GSPPEGLRAVLQREGPAGFARALRGHRGLLLXDTTFRDAHQSLLATRVRT
GPPPAGFRDILLREGPEGFARAVRNHQGLLLMDTTFRDAHQSLLATRVRT
VPATKGWKQILDSEGPEAFARAVRANKGCLIMDTTWRDAHQSLLATRVRT
-APPPGSKQRLTKLGPEGFALWMRESKTVGVTDTTFRDAHQSLLATRVRT
--------------------------------------------------

584
584
584
599
545

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

HDLKKIAPYVAHNFSKLFSMENWGGATFDVAMRFLYECPWRRLQELRELI
RDLARIAPFVAHSLSPLCSMETWGGATFDVAMRFLHECPWERLRELRRLV
HDLKKIAPYVAHNFNNLFSIENWGGATFDVAMRFLYECPWRRLQELRELI
IDLLNIAHETSHALANAYSLECWGGATFDVAMRFLYEDPWDRLRKLRKAV
SGLMRVAPYIARMTPELLSVECWGGATYDVALRFLKQDPWDRLAALREAL
--------------------------------------------------

634
634
634
649
595

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

PNIRFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDVFRVFDSLNYLP 684
PNIPFQMLLRGANAVGYTNYPDNVIYRFCEVAAANGMDIFRIFDALNYLP 684
PNIPFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDVFRIFDSLNYLP 684
PNIPFQMLLRGANGVAYSSLPDNAIYHFCKQAKKCGVDIFRVFDALNDVD 699
PNICLQMLLRGRNTVGYTPYPEQVTSAFVEEAAQTGVDIFRIFDSLNNIV 645
-------------------------------------------------gi|
NMLLGMEAAGSAGG-VVEAAISYTGDVADPSRTKYSLQYYMGLAEALVRA 733
NLLLGVEAVGRAGA-VVEAALSYTGDVADPTRTKYSLDYYLGLAKELVAA 733
NMLLGMEAAGSAGG-VVEAAISYTGDVADPSRTKYSLEYYMGLAEELVRA 733
QLEVGIKAVHAAEG-VVEATICYSGDMLNPSK-KYNLPYYLDLVDKVVQF 747
AMRPAIDAVLNTGTTIAEVAMSYTGDLSDPGEDLYTLDYYLRLAEAIVDA 695
--------------------------------------------------

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

GTHILCIKDMAGLLKPTACTMLVSSLRDRFPDLPLHIHTHAPSGAGVAAM
GTHILCIKDMAGLLTPAAARLLVSSLRDRFPDVPIHVHTHDTAGAAIATL
GTHILCIKDMAGLLKPAACTMLVSSLRDRFPDLPLHIHTHDTSGSGVAAM
KPHVLGIKDMAGVLKPQAARLLIGSIRERYPDLPIHVHTHDSAGTGVASM
GAHVLAIKDMAGLLRAPAAATLVAALKSRF-DLPVHVHTHDTPGGQLATY
--------------------------------------------------

783
783
783
797
744

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.

LACAQAGADVVDVAADSMSGMTSQPSMGALVACTRGTPLDTEVPMERVFD
LAAANADADVVDVAVDAMSGMTSQPSMGALVACARGTPLDTGIALERVFE
LACAQAGADVVDVAVDSMSGMTSQPSMGALVACTKGTPLDTEVPLERVFD
IACAQAGADAVDAATDSLSGMTSQPSIGAILASLEGTEHDPGLNSAQVRA
AAAWAAGADAVDGAAAPLSGTTSQPSLSSIVAAAARTEFDTGLSLSAVCD

833
833
833
847
794

gi|145175|gb|AAA23409.1|

--------------------------------------------------

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

YSEYWEGARGLYAAFDCTATMKSGNSDVYENEIPGGQYTNLHFQAHSMGL
YSEYWEGARGLYAAFDCTATMKSGNADVYENEIPGGQYTNLHFQAHAMGL
YSEYWEGARGLYAAFDCTATMKSGNSDVYENEIPGGQYTNLHFQAHSMGL
LDTYWAQLRLLYSPFEAGLTGP--DPEVYEHEIPGGQLTNLIFQASQLGL
LEPYWEALRKVYAPFESGLAAP--TGRVYTHEIPGGQLSNLRQQAIALGL
--------------------------------------------------

883
883
883
895
842

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

GSKFKEVKKAYVEANQMLGDLIKVTPSSKIVGDLAQFMVQNGLSRAEAEA
GHKFKEVKKAYAEANKLLGDLIKVTPSSKVVGDLAQFMVQNGLSREEAEA
GSKFKEVKKAYVEANQMLGDLIKVTPSSKIVGDLAQFMVQNGLSRAEAEA
GQQWAETKKAYESANDLLGDVVKVTPTSKVVGDLAQFMVSNKLTAEDVIA
GDRFEDVENAYAGADRVLGRLVKVTPSSKVVGDLALALVGAGASAEEFAA
--------------------------------------------------

933
933
933
945
892

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

QAEELSFPRSVVEFLQGYIGVPHGGFPEPFRSKVLKDLPRVEGRPGASLP
RADELSFPLSVVEFLQGYIGTPPGGFPEPFRSKVLKDLPRVEGRPGASLP
QAEELSFPRSVVEFLQGYIGIPHGGFPEPFRSKVLKDLPRIEGRPGASLP
RAGELDFPGSVLEFLEGLMGQPYGGFPEPLRSRALRDRRKLDKRPGLYLE
EPGLYDLPDSVIGFLRGELGDPPGGWPEPLRTKALDGR-------GPAKP
--------------------------------------------------

983
983
983
995
935

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

PLDLQALEKELVDRHGEEVTPEDVLSAAMYPDVFAHFKDFTATFGPLDSL
PLDFEALSQELGARDGTPPSPEDLLSAALYPKVYAEFRDFTSTFGPVSCL
PLNLKELEKDLIDRHGEEVTPEDVLSAAMYPDVFAQFKDFTATFGPLDSL
PLDLAKIKSQIRENYG-AATEYDVASYAMYPKVFEDYKKFVAKFGDLSVL
EQELTVEQEAVLAAPG--PKRRAMLNHLLFAGPTAEFEAHREEFGDTSRL
--------------------------------------------------

1033
1033
1033
1044
983

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

NTRLFLQGPKIAEEFEVELERGKTLHIKALAVSDLN-RAGQRQVFFELNG
GTRLFLEGPTIAEEFEVELERGKTLHIKALALGDLN-AAGQREAFFELNG
NTRLFLQGPKIAEEFEVELERGKTLHIKALAVSDLN-RAGQRQVFFELNG
PTRYFLAKPEIGEEFHVELEKGKVLILKLLAIGPLSEQTGQREVFYEVNG
SANQFFYGLRHGEEHRVTLEPGVELLIGLEAISDAD-ERGMRTVMCIING
--------------------------------------------------

1082
1082
1082
1094
1032

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

QLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPGKVIDIKVVAGAKVA
QLRSILVRDTQALKEMHVHPKADRSAKGQVGAPMPGEVVEVRVKEGEAVE
QLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPGKVIDVKVAAGAKVV
EVRQVSVDDKKASVENTARPKAELGDSSQVGAPMSGVVVEIRVHDGLEVK
QLRPVMVRDRSIASDIPVSERADKTNPDHVAAPFAG-VVTVTVAQGDAVE
--------------------------------------------------

1132
1132
1132
1144
1081

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|

KGQPLCVLSAMKMETVVTSPMEGTVRKVHVTKDMTLEGDDLILEIE-KGAPLCVLSAMKMETVVTAPRGGTVSRLHVRPGMSLEGDDLIAEIE-KGQPLCVLSAMKMETVVTSPMEGTIRKVHVTKDMTLEGDDLILEIE-KGDPIAVLSAMKMEMVISAPHSGKVSSLLVKEGDSVDGQDLVCKIVKA
AGQTLATIEAMKMEAAITATKAGTLNRIAVAATAQVESGDLLMVLT-------------------------------------------------

Totally Conserved Region

Cys
C230, C337

Lys
K4, K116, K159, K238, K387

1178
1178
1178
1192
1127

Qn 2
Cysteine to Lysine
Cystine

C230

C337

Lysine
K4
K116
K159
K238
K387
K4
K116
K159
K238
K387

Distance between nitrogen amino side group to sulphur

[Armstrong]
35.91
15.95
16.31
4.60
12.57
37.89
26.38
27.50
15.05
12.80

Table 1: Distance between nitrogen amino side group to sulphur [Armstrong]

Fig 1: Distance between nitrogen amino side group to sulphur for CYS230

Fig 2: Distance between nitrogen amino side group to sulphur for CYS337

Based on the table 1 comparison, the Cys 230 side chain sulphur atom is the closest
at 4.6 from the e-amino atom of Lys238 and would allow the formation of an ion
pair.

Qn 3
Residues within 4 armstrong distance of Lysine 238
HIS209, GLU296, GLU211, GLU276 (Refer to Figure 3)
Amino side chain of lysine is positively charged and can only form ion pair with
negatively charged side chain this makes Glutamic amino acids the most likely
candidate
Characteristic of Amino Acid;
1. Lysine Positively charged, Basic and Polar
2. Glutamic Acid Negatively Charged, Acidic and Polar

3. Histidine Positively charged, Basic and Polar

Potential Pair with
Lysine238
GLU211
GLU276
GLU296

Distance to Carbon A

Distance to Carbon B

3.37
3.28
5.10

4.38
5.00
3.22

Table2. amino acids within 4 armstrong distance from Lysine 238

Fig 3. Distance observed from Lys238 to the carbons of amino acids within 4
armstrong distance.
Potential Pair
GLU296 has the shortest distance of 3.22 to the side chain of Lysine 238 therefore it
is likely to form an ion pair with Lysine 238.

Qn 4

Fig 4: Distance of His236 to 1N of the biotin and Ureido Oxygen of the biotin
The side chain of histidine contains an imidazole ring which contains a nitrogen
atom with lone pair electrons in the sp2 orbital, this electron pair can be donated
to a hydrogen atom on 1-N of biotin to form a new bond and the resultant
negative charge on the ureido oxygen of the enolised biotin is stabilized by the
positively charged nitrogen atom formed on histidine.

Qn 5
To test if Histidine(His236) and Lysine(Glu296)is important for the biotin
carboxylation reaction, the residues would need to be mutated first with site
directed mutagenesis. The site directed mutagenesis reaction would be carried out
using polymerase chain reaction (PCR) with overlapping extension using
mutagenic internal primers to produce separate mutants H236A (histidine to
alanine) and E296Q (glutamate to glutatmine). This would be followed with
sequencing of entire mutant gene to confirm that the desired mutation was made
and no other mutations had been incorporated. Once correct identity is confirmed
the biotin carboxylase enzyme would be overexpressed and purified using affinity
chromatography for enzymatic assay.
First, the enzymatic rate of ATP hydrolysis to ADP is measured for both mutant and
wild-type in the presence of biotin by coupling the production of ADP to pyruvate
kinase and lactate dehydrogenase for oxidation of NADH to NAD+. The oxidation
of NADH would be monitored with a spectrophotometer measuring absorbance at
340 nm and would be an indication of the ADP production.
14C fixation assay is then used to determine the amount of carboxybiotin
produced by biotin carboxylase. The result will then be compared with the
enzymatic assay for production of ADP from the ATP hydrolysis assay to determine
the stoichiometric formation of ADP and carboxybiotin. In order for biotin
carboxylation to be uncoupled with ATP hydrolysis the stoichiometric ratio for
carboxybiotin to ADP production must be >1.This would indicate that biotin
carboxylation occurred faster than the formation of ADP and signal uncoupling . If
the ratio is 1:1 the carboxyl transfer step had not been uncoupled from the
hydrolysis of ATP. The stoichiometric result is then compared with the wild type
enzyme to confirm its validity.

Source:
Sloane V, Blanchard CZ, Guillot F, Waldrop GL. Site-directed mutagenesis of ATP
binding residues of biotin carboxylase. Insight into the mechanism of catalysis.J
Biol Chem. 2001 Jul 6;276(27):24991-6. Epub 2001 May 9.

Sloane V, Waldrop GL. Kinetic characterization of mutations found in propionic

acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin
carboxylase. J Biol Chem. 2004 Apr 16;279(16):15772-8. Epub 2004 Feb 11.

Qn 6
ClustalW multiple sequence alignment (between pyruvate carboxylase of 5
organisms, biotin carboxylase of Escherichia coli and carbamoyl phosphate
synthase large subunit of Escherichia coli APEC O1
gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

MLKFRTVH---GGLRLLGIRRTSTAPAASPNVRR-LEYKPIKKVMVANRG
MSQLCVPR---GGRALLGAWRLPLLRPPPGSVRS-ASCQPIRKVLVANRG
MLKFQTVR---GGLRLLGVRRSSTAPVASPNVRR-LEYKPIKKVMVANRG
MAAPRQPEEAVDDTEFIDDHHDQHRDSVHTRLRANSAIMQFQKILVANRG
---------------------------------------MFSKVLVANRG
---------------------------------------MLDKIVIANRG
----------------------------------MPKRTDIKSILILGAG
: .::: . *

46
46
46
50
11
11
16

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

EIAIRVFRACTELGIRTVAIYSEQDTGQMHRQKADEAYLIG-RG-LAPVQ
EIAIRVFRACTELGLRTVAVYSEQDTGQMHRQKADEAYLVG-RG-LPPVQ
EIAIRVFRACTELGIRTVAVYSEQDTGQMHRQKADEAYLIG-RG-LAPVQ
EIPIRIFRTAHELSLQTVAVYSHEDHLSMHRQKADEAYMIGKRGQYTPVG
EIAIRAFRAVYELGAATVAVYPYEDRNSLHRSKADESYQIGVEG--HPVR
EIALRILRACKELGIKTVAVHSSADRDLKHVLLADETVCIGPAP---SVK
PIVIGQACEFDYSGAQACKALREEGYRVILVNSNPATIMTDPEMADATYI
* :
. :
.
:
.
.

94
94
94
100
59
58
66

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

AYLHIPDIIKVAKENNVDAVHPGYG-----FLSERADFAQACQDAGVRFI
AYLHVPDIIRVARENAVDAIHPGYG-----FLSERADFAQACVDAGVRFV
AYLHIPDIIKVAKENGVDAVHPGYG-----FLSERADFAQACQDAGVRFI
AYLAIDEIVKIALEHGVHLIHPGYG-----FLSENAEFARKVEQSGMVFV
AYLNVDHIVSTALDCGADAIYPGYG-----FLSENPELATACAAAGITFV
SYLNIPAIISAAEITGAVAIHPGYG-----FLSENANFAEQVERSGFIFI
EPIHWEVVRKIIEKERPDAVLPTMGGQTALNCALELERQGVLEEFGVTMI
:
:
: * *
: . :
*. ::

139
139
139
145
104
103
116

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

GPSPEVVRKMGDKVEARAIAIAAGVPVVPGTDAPITSLHEAH-EFSNTYG
GPPPEVVRKMGDKVEARSIAIAAGVPVVPGTSAPVATLGEAQ-DFAARVG
GPSPEVVRKMGDKVEARAIAIAAGVPVVPGTNSPINSLHEAH-EFSNTYG
GPTPQTIESLGDKVSARQLAIRCDVPVVPGTPGPVERYEEVK-AFTDTYG
GPSADVLELTGNKARAIAAARAAGLPVLASS-QPSADVHELV-AAAQSME
GPKAETIRLMGDKVSAIAAMKKAGVPCVPGSDGPLGDDMDKNRAIAKRIG
GATADAIDKAEDRRRFDVAMKKIGLETARSG---IAHSMEEALAVAAEVG
*. .:.:
::
.:
.
:
:

188
188
188
194
152
153
163

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

FPIIFKAAYGGGGRGMRVVHSYEELEENYTRAYSEAWPAFGNGALFVEKF
FPIIFKAAHGGGGRGMRAVRGPQELEESFSRASSEALAAFGDGALFVEKL
FPIIFKAAYGGGGRGMRVVHSYEELEENYTRAYSEALAAFGNGALFVEKF
FPIIIKAAFGGGGRGMRVVRDQAELRDSFERATSEARSAFGNGTVFVERF
FPLFVKAVAGGGGRGMRRVAAAADLQEAIEAASREAESAFGDATVFLEQA
YPVIIKASGGGGGRGMRVVRGDAELAQSISMTRAEAKAAFSNDMVYMEKY
FPCIIRPSFTMGGSGGGIAYNREEFEEICARG----LDLSPTKELLIDES
:* :.:.
** *
.
:: :
: ::.

238
238
238
244
202
203
209

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

IEKPRHIEVQILGDQYGNILHLYERD--CSIQRRHQKVVEIAPAAHLDPQ
MERPRHIEGQILGDKHGNVVHLYERD--CSIQRRHQKVVEIAPAARLDPQ
IEKPRHIEVQILGDQYGNILHLYERD--CSIQRRHQKVVEIAPATHLDPQ
LDRPKHIEVQLLGDNHGNVVHLFERD--CSVQRRHQKVVEIAPAKDLPAD
VVNPRHIEVQILADGSGEVIHLFERD--CSVQRRHQKVIELAPAPNLEPA
LENPRHVEIQVLADGQGNAIYLAERD--CSMQRRHQKVVEEAPAPGITPE
LIGWKEYEMEVVRDKNDNCIIVCSIENFDAMGIHTGDSITVAPAQTLTDK
:
:. * ::: * .: : : . :
:: : . : *** :

286
286
286
292
250
251
259

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

LRTRLTSDSVKLAKQVGYENAG-TVEFLVDR-HGKHYFIEVNSRLQVEHT
LRAQLASDAVRIAQQVGYENAG-TVEFLVDR-DGKHYFIEVNSRLQVEHT
LRSRLTSDSVKLAKQVGYENAG-TVEFLVDK-HGKHYFIEVNSRLQVEHT
VRDRILADAVKLAKSVNYRNAG-TAEFLVDQ-QNRYYFIEINPRIQVEHT
LRARICDDAVAFARQIGYSCAG-TVEFLLDE-RGQHVFIEMNPRIQVEHT
LRRYIGERCAKACVDIGYRGAG-TFEFLFE--NGEFYFIEMNTRIQVEHP
EYQIMRNASMAVLREIGVETGGSNVQFAVNPKNGRLIVIEMNPRVSRSSA
:
.
.:.
.* . :* .:
.. .**:*.*:. . .

334
334
334
340
298
298
309

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

VTEEITDVDLVHAQIHVAEGRSLPDLGLRQENIRING--CAIQCRVTTED
VTEEITGVDLVQAQLLVAAGRSLSELGLQQDSVRVNG--CAIQCRVTTED
VTEEITDVDLVHAQIHVSEGRSLPDLGLRQENIRING--CAIQCRVTTED
ITEEITGIDIVAAQIQIAAGATLEQLGLTQDRISTRG--FAIQCRITTED
VTEEITDVDLVSAQLRIAAGETLADLGLAQDTLQIHG--AAIQCRITTED
VTEMITGVDLIKEQLRIAAG---QPLSIKQEEVHVRG--HAVECRINAED
LASKATGFPIAKVAAKLAVGYTLDELMNDITGGRTPASFEPSIDYVVTKI
::. *.. :
:: *
*
.
.
: ::

382
382
382
388
346
343
359

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

PAPTFQPDTGRIEVFRSGEGMGIRLDNASAFQGAVISPHYDSLLVKVIAH
PARGFQPDTGRIEVFRSGEGMGIRLDGASAFQGALISPHYDSLLVKVIAH
PARSFQPDTGRIEVFRSGEGMGIRLDNASAFQGAVISPHYDSLLVKVIAH
PSKGFSPDTGKIEVYRSAGGNGVRLDGGNGFAGAIITPHYDSMLVKCTCR
PANGFRPGTGRITGYRSPGGAGIRLDGGTNIG-AEVTAHFDSMLVKLSCR
P-NTFLPSPGKITRFHAPGGFGVRWES-HIYAGYTVPPYYDSMIGKLICY
PRFNFEKFAGANDRLTTQMKSVGEVMAIGRTQQESLQKALRGLEVGATGF
*
*
.*
:
.
:
.:

432
432
432
438
395
391
409

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

GKDHPTAATKMSRALAEFRVRGVKTNIAFLQNVLNNQQFLAGTVDTQFID
GPDQPSAAAKMSRALGEFRIRGVKTNIPFLQNVLAHPQFLGGAVDTQFID
GKDHPTAATKMSRALAEFRVRGVKTNIPFLQNVLNNQQFLAGIVDTQFID
GSTYEIARRKVVRALVEFRIRGVKTNIPFLTSLLSHPVFVDGTCWTTFID
GRDFDTAVRRARRAVAEFRIRGVSTNIPFLQAVLDDPDFQSGRVTTSFID
GENRDVAIARMKNALQELIIDGIKTNVDLQIRIMNDENFQHGGTNIHYLE
DPKVSLDDPEALTKIRRELKDAGAERIWYIADAFRAGLSVDGVFNLTNID
.
.
: .
.
.:
:
*
::

482
482
482
488
445
441
459

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

ENPDVFQLRPAQNRAQKLLHYLGHVMVNGPTTP---IPVKASPSPTDPVV
ENPELFHLRPSQNRAQKLLHYLGHVMVNGPSTP---LPVKAKAAVVEPVP
ENPELFQLRPAQNRAQKLLHYLGHVMVNGPTTP---IPVKVSPSPVDPIV
DTPELFALVGSQNRAQKLLAYLGDVAVNGSSIKGQIGEPKLKGDIIKPVL
ERPGLLTARSSADRGTKILNYLADVTVNQPHGP---RPSAVYPRDKLPVI
KKLGLQEK-----------------------------------------R--WFLVQIEELVRLEEKVAEVGITGLHAEFLR----------------.

529
529
529
538
492
449
490

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

PAVP------IGPPPAGFRDILLREGPEGFARAVRNHPGLLLMDTTFRDA
PPVP------MGSPPEGLRAVLQREGPAGFARALRGHRGLLLXDTTFRDA
PVVP------IGPPPAGFRDILLREGPEGFARAVRNHQGLLLMDTTFRDA
HDAAGKPLDVSVPATKGWKQILDSEGPEAFARAVRANKGCLIMDTTWRDA
DLTA--------APPPGSKQRLTKLGPEGFALWMRESKTVGVTDTTFRDA
-----------------------------------------------------------------------------------------QLKRKGFADA

573
573
573
588
534

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

HQSLLATRVRTHDLKKIAPYVAHNFSKLFSMENWGGATFDVAMRFLYECP
HQSLLATRVRTRDLARIAPFVAHSLSPLCSMETWGGATFDVAMRFLHECP
HQSLLATRVRTHDLKKIAPYVAHNFNNLFSIENWGGATFDVAMRFLYECP
HQSLLATRVRTIDLLNIAHETSHALANAYSLECWGGATFDVAMRFLYEDP
HQSLLATRVRTSGLMRVAPYIARMTPELLSVECWGGATYDVALRFLKQDP
-------------------------------------------------RLAKLAG-VREAEIRKLR--DQYDLHPVYKRVDTCAAEFATDTAYMYS-T

623
623
623
638
584

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

WRRLQELRELIPNIRFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDV
WERLRELRRLVPNIPFQMLLRGANAVGYTNYPDNVIYRFCEVAAANGMDI
WRRLQELRELIPNIPFQMLLRGANAVGYTNYPDNVVFKFCEVAKENGMDV
WDRLRKLRKAVPNIPFQMLLRGANGVAYSSLPDNAIYHFCKQAKKCGVDI
WDRLAALREALPNICLQMLLRGRNTVGYTPYPEQVTSAFVEEAAQTGVDI
-------------------------------------------------YEEECEANPSTDREKIMVLGGGPNRIGQGIEFDYCCVHASLALREDGYET

673
673
673
688
634

500

546

596

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

FRVFDSLNYLPNMLLGMEAAGSAGG-VVEAAISYTGDVADPSRTKYSLQY
FRIFDALNYLPNLLLGVEAVGRAGA-VVEAALSYTGDVADPTRTKYSLDY
FRIFDSLNYLPNMLLGMEAAGSAGG-VVEAAISYTGDVADPSRTKYSLEY
FRVFDALNDVDQLEVGIKAVHAAEG-VVEATICYSGDMLNPSK-KYNLPY
FRIFDSLNNIVAMRPAIDAVLNTGTTIAEVAMSYTGDLSDPGEDLYTLDY
-------------------------------------------------IMVNCNPETVSTDYDTSDRLYFEPVTLEDVLEIVRIEKPKGVIVQYGGQT

722
722
722
736
684

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

YMGLAEALVRAGTHILCIKDMAGLLKPTACTMLVSSLRDRFPDLPLHIHT
YLGLAKELVAAGTHILCIKDMAGLLTPAAARLLVSSLRDRFPDVPIHVHT
YMGLAEELVRAGTHILCIKDMAGLLKPAACTMLVSSLRDRFPDLPLHIHT
YLDLVDKVVQFKPHVLGIKDMAGVLKPQAARLLIGSIRERYPDLPIHVHT
YLRLAEAIVDAGAHVLAIKDMAGLLRAPAAATLVAALKSRF-DLPVHVHT
-------------------------------------------------PLKLARALEAAG---------VPVIGTSPDAIDRAEDRERFQHAVERLKL

772
772
772
786
733

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

HAPSGAGVAAMLACAQAGADVVDVAADSMSGMTSQPSMGALVACTRG--HDTAGAAIATLLAAANADADVVDVAVDAMSGMTSQPSMGALVACARG--HDTSGSGVAAMLACAQAGADVVDVAVDSMSGMTSQPSMGALVACTKG--HDSAGTGVASMIACAQAGADAVDAATDSLSGMTSQPSIGAILASLEG--HDTPGGQLATYAAAWAAGADAVDGAAAPLSGTTSQPSLSSIVAAAAR---------------------------------------------------KQPANATVTTIEMAVEKAKEIGYPLVVRPSYVLGGRAMEIVYDEADLRRY

819
819
819
833
780

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

TPLDTEVPMERVFDYSEYWEGARGLYAAFDCTATMKSGNSDVYENEIPGG
TPLDTGIALERVFEYSEYWEGARGLYAAFDCTATMKSGNADVYENEIPGG
TPLDTEVPLERVFDYSEYWEGARGLYAAFDCTATMKSGNSDVYENEIPGG
TEHDPGLNSAQVRALDTYWAQLRLLYSPFEAGLTGP--DPEVYEHEIPGG
TEFDTGLSLSAVCDLEPYWEALRKVYAPFESGLAAP--TGRVYTHEIPGG
-------------------------------------------------FQTAVSVSNDAPVLLDHFLDDAVEVDVDAICDGEMVLIGGIMEHIEQAGV

869
869
869
881
828

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

QYTNLHFQAHSMGLGSKFKEVKKAYVEANQMLGDLIKVTPSSKIVGDLAQ
QYTNLHFQAHAMGLGHKFKEVKKAYAEANKLLGDLIKVTPSSKVVGDLAQ
QYTNLHFQAHSMGLGSKFKEVKKAYVEANQMLGDLIKVTPSSKIVGDLAQ
QLTNLIFQASQLGLGQQWAETKKAYESANDLLGDVVKVTPTSKVVGDLAQ
QLSNLRQQAIALGLGDRFEDVENAYAGADRVLGRLVKVTPSSKVVGDLAL
-------------------------------------------------HSGDSACSLPAYTLSQEIQDVMRQQVQKLAFELQVRGLMNVQFAVKNNEV

919
919
919
931
878

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

FMVQNGLSRAEAEAQAEELSFPRSVVEFLQGYIGVPHGGFPEPFRSKVLK
FMVQNGLSREEAEARADELSFPLSVVEFLQGYIGTPPGGFPEPFRSKVLK
FMVQNGLSRAEAEAQAEELSFPRSVVEFLQGYIGIPHGGFPEPFRSKVLK
FMVSNKLTAEDVIARAGELDFPGSVLEFLEGLMGQPYGGFPEPLRSRALR
ALVGAGASAEEFAAEPGLYDLPDSVIGFLRGELGDPPGGWPEPLRTKALD
-------------------------------------------------YLIEVNPR-------------AARTVPFVSKATGVPLAKVAARVMAG---

969
969
969
981
928

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

DLPRVEGRPGASLPPLDLQALEKELVDRHGEEVTPEDVLSAAMYPDVFAH
DLPRVEGRPGASLPPLDFEALSQELGARDGTPPSPEDLLSAALYPKVYAE
DLPRIEGRPGASLPPLNLKELEKDLIDRHGEEVTPEDVLSAAMYPDVFAQ
DRRKLDKRPGLYLEPLDLAKIKSQIRENYG-AATEYDVASYAMYPKVFED
GR-------GPAKPEQELTVEQEAVLAAPG--PKRRAMLNHLLFAGPTAE
-------------------------------------------------------------KSLAEQGVTKEVIPPYYSVKEVVLPFNKFPGVDPLLGP

1019
1019
1019
1030
969

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

FKDFTATFGPLDSLNTRLFLQGPKIAEEFEVELERGKTLHIKALAVSDLN
FRDFTSTFGPVSCLGTRLFLEGPTIAEEFEVELERGKTLHIKALALGDLN
FKDFTATFGPLDSLNTRLFLQGPKIAEEFEVELERGKTLHIKALAVSDLN
YKKFVAKFGDLSVLPTRYFLAKPEIGEEFHVELEKGKVLILKLLAIGPLS
FEAHREEFGDTSRLSANQFFYGLRHGEEHRVTLEPGVELLIGLEAISDAD
-------------------------------------------------EMRSTGEVMGVGRTFAEAFAKAQLGSNSTMKKHGRALLSVREGDKERVVD

1069
1069
1069
1080
1019

646

687

737

787

837

871

909

959

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

-RAGQRQVFFELNGQLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPG
-AAGQREAFFELNGQLRSILVRDTQALKEMHVHPKADRSAKGQVGAPMPG
-RAGQRQVFFELNGQLRSILVKDTQAMKEMHFHPKALKDVKGQIGAPMPG
EQTGQREVFYEVNGEVRQVSVDDKKASVENTARPKAELGDSSQVGAPMSG
-ERGMRTVMCIINGQLRPVMVRDRSIASDIPVSERADKTNPDHVAAPFAG
-------------------------------------------------LAAKLLKQGFELDATHGTAIVLGEAGINPRLVNKVHEGRPHIQDRIKNGE

1118
1118
1118
1130
1068

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

KVIDIKVVAGAKVAKGQPLCVLSAMKMETVVTSPMEGTVRKVHVTKDMTL
EVVEVRVKEGEAVEKGAPLCVLSAMKMETVVTAPRGGTVSRLHVRPGMSL
KVIDVKVAAGAKVVKGQPLCVLSAMKMETVVTSPMEGTIRKVHVTKDMTL
VVVEIRVHDGLEVKKGDPIAVLSAMKMEMVISAPHSGKVSSLLVKEGDSV
-VVTVTVAQGDAVEAGQTLATIEAMKMEAAITATKAGTLNRIAVAATAQV
-------------------------------------------------YTYIINTTSGRRAIEDSRVIRRSALQYKVHYDTTLNGGFATAMALNADAT

1168
1168
1168
1180
1117

gi|632808|gb|AAB31500.1|
gi|45383466|ref|NP_989677.1|
gi|1040974|gb|AAC52668.1|
gi|12044690|emb|CAC19838.1|
gi|504618726|ref|WP_014805828.
gi|145175|gb|AAA23409.1|
gi|117622323|ref|YP_851236.1|

EGDDLILEIE---EGDDLIAEIE---EGDDLILEIE---DGQDLVCKIVKA-ESGDLLMVLT----------------EKVISVQEMHAQIK

1009

1059

1178
1178
1178
1192
1127
1073

Conserved residues (labeled with an (*) red words) and partially conserved
residues (labelled with an (:) blue words), amino acids highlighted in yellow based
on residues shown in picture below

Fig. 2B in Role of Conserved Residues within the Carboxy Phosphate Domain of

Carbamoyl Phosphate Synthetase (Stapleton et al. 1996, p. 14357).

The table below (Table 3) shows the residues of interest of the enzymes which are
shown in
YP_85123
6
Carbamoy
l
Phosphat
e
Synthase
Arg82

AAA23409.
1
Biotin
Carboxylas
e

Pyruvate
Carboxylas
e

Chemical
properties
Similarities

Structure
Similariti
es

Conservati
on of
amino acid
residue

Hydroge
n
Bonding

Glycine

NA

NA

No

NA

Arg129

Lysine

Gly, Asn,
Ala
Lysine

Long
side
chain

Partially

Donor

Similar
side
chain
carbony
l group
with
one less
carbon
NA

Partially

NA

No

NA

NA

No

Accepto
r to
donor

NA

Partially

3
carbon
side
chain

Partially

Donor
NH2 to
OH
No

Same
amino
acid

Fully

NA

Same

Fully

NA

Asp207

Glutamat
e

Glutamat
e

Glu215

Histidine

Histidine

His243

Arginine

Arginine

Asn283

Threonin
e

Threonin
e

Gln285

Glutamat
e

Glutamat
e

Hydrophili
c
Positive
Charged
Basic
Polar
Hydrophil
ic
Polar

Glu299

Glutamat
e

Glutamat
e

Asn301

Asparagi

Asparagi

Positive
charged
basic
polar
Hydroph
ilic
Negativ
e
Charged
Acidic
Polar
Hydroph
ilic

Hydroph
ilic
Polar
carbonyl
oxygen
Neutral
Charged
Polar
Hydroph
ilic
Neutral

Arg303

ne

ne

Arginine

Arginine

Charged
Polar
Positive
Charged
Basic
Polar

amino
acid
Same
amino
acid

Fully

Donor

Table 3. Summary Comparison of properties of the amino acid side chains in the
same region