EzCatDB: S00239
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DB codeS00239
RLCP classification6.10.398000.111 : Double-bonded atom exchange
6.10.431300.120 : Double-bonded atom exchange
8.121.1440000.6450 : Isomerization
4.202.112900.6000 : Addition
5.200.552010.6500 : Elimination
5.1202.1504200.6501 : Elimination
8.11121.166400.120 : Isomerization
CATH domainDomain 13.20.20.70 : TIM BarrelCatalytic domain
E.C.4.2.1.24

CATH domainRelated DB codes (homologues)
3.20.20.70 : TIM BarrelS00215,S00217,S00218,S00219,S00532,S00198,S00220,S00745,S00537,S00538,S00539,S00826,S00841,S00235,S00240,S00243,S00244,S00199,S00200,S00201,S00221,S00222,S00847,S00224,S00225,S00226,D00014,D00029,M00141,T00015,T00239,D00664,D00665,D00804,D00863,T00089

Enzyme Name
UniProtKBKEGG

P0ACB2
Protein nameDelta-aminolevulinic acid dehydrataseporphobilinogen synthase
aminolevulinate dehydratase
delta-aminolevulinate dehydratase
delta-aminolevulinic acid dehydrase
delta-aminolevulinic acid dehydratase
aminolevulinic dehydratase
delta-aminolevulinic dehydratase
5-levulinic acid dehydratase
5-aminolevulinate hydro-lyase (adding 5-aminolevulinate andcyclizing)
SynonymsALADH
ALAD
EC 4.2.1.24
Porphobilinogen synthase
RefSeqNP_414903.4 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_488662.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PfamPF00490 (ALAD)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00860Porphyrin and chlorophyll metabolism

UniProtKB:Accession NumberP0ACB2
Entry nameHEM2_ECOLI
Activity2 5-aminolevulinate = porphobilinogen + 2 H(2)O.
SubunitHomooctamer.
Subcellular location
CofactorBinds 1 magnesium ion per monomer.,Binds 1 zinc ion per monomer.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00038C00305C00430C00931C00001






CompoundZincMagnesium5-AminolevulinatePorphobilinogenH2OP-side ALA carbinolamine transition-stateP-side ALA Schiff-base intermediateA-side ALA carbinolamine + P-side ALA Schiff-base intermediateA-side/P-side ALA Schiff-base intermediateA-side enamine + P-side Schiff-base intermediateC-C bonded intermediateC-N bonded intermediate
Typeheavy metaldivalent metal (Ca2+, Mg2+)amino acids,carbohydrateamine group,aromatic ring (with nitrogen atoms),carboxyl groupH2O






ChEBI29105
18420
17549
356416
17381
15377







PubChem32051
888
7048523
137
1021
962
22247451







                    
1b4eABound:_ZN 400Analogue:_ZN 401UnboundUnbound UnboundIntermediate-analogue:LEAUnboundUnboundUnboundUnboundUnbound
1i8jABound:_ZNBound:_MGUnboundUnbound UnboundUnboundUnboundIntermediate-analogue:DSBUnboundUnboundUnbound
1i8jBBound:_ZNBound:_MGUnboundUnbound UnboundUnboundUnboundIntermediate-analogue:DSBUnboundUnboundUnbound
1l6sABound:_ZNBound:_MGUnboundUnbound UnboundUnboundUnboundIntermediate-analogue:DSBUnboundUnboundUnbound
1l6sBBound:_ZNBound:_MGUnboundUnbound UnboundUnboundUnboundIntermediate-analogue:DSBUnboundUnboundUnbound
1l6yABound:_ZNBound:_MGUnboundUnbound UnboundIntermediate-analogue:4OXUnboundUnboundUnboundUnboundUnbound
1l6yBBound:_ZNBound:_MGUnboundUnbound UnboundIntermediate-analogue:4OXUnboundUnboundUnboundUnboundUnbound

Active-site residues
pdbCatalytic residuesCofactor-binding residues
          
1b4eAASP 118;SER 165;LYS 195;LYS 247
CYS 120;CYS 122;CYS 130(Zinc binding);GLU 232(Magnesium binding)
1i8jAASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)
1i8jBASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)
1l6sAASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)
1l6sBASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)
1l6yAASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)
1l6yBASP 117;SER 164;LYS 194;LYS 246
CYS 119;CYS 121;CYS 129(Zinc binding);GLU 231(Magnesium binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[1]Scheme 2, p.2855
[5]Fig.1, p.8346
[7]Fig.7, p.2122
[11]Fig.1, Fig.2, p.11561
[13]Fig.3, p.169-1752
[17]p.1027
[20]Fig.7, p.4273-4275
[23]Fig.1, p.599
[24]Fig.6, p.1254
[25]p.428
[27]Fig.8, p.191-19411
[28]p.8234-8256
[29]Fig.4, p.199-2008
[31]p.138-139
[33]Scheme 2, p.589-5907
[34]p.19797-19798
[35]Fig.6, p.243-24510
[36]Scheme 2, p.736-7378
[38]p.567-568
[41]p.1225-1226
[42]Fig.5, p.8251-8252

references
[1]
PubMed ID6967019
JournalFEBS Lett
Year1980
Volume114
Pages283-6
AuthorsJordan PM, Seehra JS
Title13C NMR as a probe for the study of enzyme-catalysed reactions: mechanism of action of 5-aminolevulinic acid dehydratase.
[2]
PubMed ID3663587
JournalBiochemistry
Year1987
Volume26
Pages4258-64
AuthorsJaffe EK, Markham GD
Title13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein.
[3]
PubMed ID3166990
JournalBiochemistry
Year1988
Volume27
Pages4475-81
AuthorsJaffe EK, Markham GD
Title13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase.
[4]
PubMed ID3242540
JournalBiol Chem Hoppe Seyler
Year1988
Volume369
Pages1099-103
AuthorsPilz I, Schwarz E, Vuga M, Beyersmann D
TitleSmall angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase).
[5]
PubMed ID2252894
JournalBiochemistry
Year1990
Volume29
Pages8345-50
AuthorsJaffe EK, Markham GD, Rajagopalan JS
Title15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate.
[6]
PubMed ID1965291
JournalBiol Chem Hoppe Seyler
Year1990
Volume371
Pages1145-52
AuthorsBlock C, Lohmann RD, Beyersmann D
TitleProbing of active site residues of the zinc enzyme 5-aminolevulinate dehydratase by spin and fluorescence labels.
[7]
PubMed ID1346974
JournalBiochemistry
Year1992
Volume31
Pages2113-23
AuthorsJaffe EK, Abrams WR, Kaempfen HX, Harris KA Jr
Title5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc.
[8]
PubMed ID8424649
JournalArch Biochem Biophys
Year1993
Volume300
Pages169-77
AuthorsMitchell LW, Jaffe EK
TitlePorphobilinogen synthase from Escherichia coli is a Zn(II) metalloenzyme stimulated by Mg(II).
[9]
CommentsCHARACTERIZATION, AND SEQUENCE OF 1-5; 95-134 AND 237-253.
Medline ID93176130
PubMed ID8439296
JournalBiochem J
Year1993
Volume290
Pages279-87
AuthorsSpencer P, Jordan PM
TitlePurification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain.
Related UniProtKBP15002
[10]
PubMed ID8382991
JournalProtein Sci
Year1993
Volume2
Pages71-9
AuthorsMarkham GD, Myers CB, Harris KA Jr, Volin M, Jaffe EK
TitleSpatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.
[11]
PubMed ID7918369
JournalBiochemistry
Year1994
Volume33
Pages11554-62
AuthorsJaffe EK, Volin M, Myers CB, Abrams WR
Title5-Chloro[1,4-13C]levulinic acid modification of mammalian and bacterial porphobilinogen synthase suggests an active site containing two Zn(II).
[12]
PubMed ID7819203
JournalBiochemistry
Year1995
Volume34
Pages244-51
AuthorsJaffe EK, Ali S, Mitchell LW, Taylor KM, Volin M, Markham GD
TitleCharacterization of the role of the stimulatory magnesium of Escherichia coli porphobilinogen synthase.
[13]
PubMed ID7592564
JournalJ Bioenerg Biomembr
Year1995
Volume27
Pages169-79
AuthorsJaffe EK
TitlePorphobilinogen synthase, the first source of heme's asymmetry.
[14]
PubMed ID7592604
JournalJ Biol Chem
Year1995
Volume270
Pages24054-9
AuthorsMitchell LW, Volin M, Jaffe EK
TitleThe phylogenetically conserved histidines of Escherichia coli porphobilinogen synthase are not required for catalysis.
[15]
PubMed ID8612634
JournalEur J Biochem
Year1996
Volume236
Pages600-8
AuthorsStolz M, Dornemann D
TitlePurification, metal cofactor, N-terminal sequence and subunit composition of a 5-aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'.
[16]
PubMed ID9341235
JournalBiochemistry
Year1997
Volume36
Pages13421-7
AuthorsPetrovich RM, Jaffe EK
TitleMagnetic resonance studies on the active site and metal centers of Bradyrhizobium japonicum porphobilinogen synthase.
[17]
CommentsX-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS).
Medline ID98069651
PubMed ID9406553
JournalNat Struct Biol
Year1997
Volume4
Pages1025-31
AuthorsErskine PT, Senior N, Awan S, Lambert R, Lewis G, Tickle IJ, Sarwar M, Spencer P, Thomas P, Warren MJ, Shoolingin-Jordan PM, Wood SP, Cooper JB
TitleX-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase.
Related PDB1aw5
Related UniProtKBP05373
[18]
PubMed ID9260292
JournalProtein Sci
Year1997
Volume6
Pages1774-6
AuthorsErskine PT, Senior N, Maignan S, Cooper J, Lambert R, Lewis G, Spencer P, Awan S, Warren M, Tickle IJ, Thomas P, Wood SP, Shoolingin-Jordan PM
TitleCrystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.
[19]
PubMed ID9766004
JournalBiochem Soc Trans
Year1998
Volume26
PagesS285
AuthorsNorton E, Sarwar M, Shoolingin-Jordan P
TitleMechanistic studies on E.coli 5-aminolaevulinic acid dehydratase.
[20]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID99211872
PubMed ID10194344
JournalBiochemistry
Year1999
Volume38
Pages4266-76
AuthorsErskine PT, Norton E, Cooper JB, Lambert R, Coker A, Lewis G, Spencer P, Sarwar M, Wood SP, Warren MJ, Shoolingin-Jordan PM
TitleX-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution.
Related PDB1b4e
Related UniProtKBP15002
[21]
PubMed ID10529243
JournalBiochemistry
Year1999
Volume38
Pages13968-75
AuthorsFrankenberg N, Heinz DW, Jahn D
TitleProduction, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.
[22]
PubMed ID10529244
JournalBiochemistry
Year1999
Volume38
Pages13976-82
AuthorsFrankenberg N, Jahn D, Jaffe EK
TitlePseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.
[23]
CommentsX-RAY CRYSTALLOGRAPHY (1.67 ANGSTROMS).
Medline ID99286289
PubMed ID10356331
JournalJ Mol Biol
Year1999
Volume289
Pages591-602
AuthorsFrankenberg N, Erskine PT, Cooper JB, Shoolingin-Jordan PM, Jahn D, Heinz DW
TitleHigh resolution crystal structure of a Mg2+-dependent porphobilinogen synthase.
Related PDB1b4k
Related UniProtKBQ59643
[24]
CommentsX-RAY CRYSTALLOGRAPHY (2.15 ANGSTROMS).
Medline ID99313446
PubMed ID10386874
JournalProtein Sci
Year1999
Volume8
Pages1250-6
AuthorsErskine PT, Newbold R, Roper J, Coker A, Warren MJ, Shoolingin-Jordan PM, Wood SP, Cooper JB
TitleThe Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid.
Related PDB1ylv
Related UniProtKBP05373
[25]
CommentsX-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS).
Medline ID20207114
PubMed ID10739915
JournalActa Crystallogr D Biol Crystallogr
Year2000
Volume56
Pages421-30
AuthorsErskine PT, Duke EM, Tickle IJ, Senior NM, Warren MJ, Cooper JB
TitleMAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites.
Related PDB1qmk,1qml,1qnv
Related UniProtKBP05373
[26]
PubMed ID10913315
JournalBiochemistry
Year2000
Volume39
Pages9018-29
AuthorsKervinen J, Dunbrack RL Jr, Litwin S, Martins J, Scarrow RC, Volin M, Yeung AT, Yoon E, Jaffe EK
TitlePorphobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions.
[27]
PubMed ID10712932
JournalChem Biol
Year2000
Volume7
Pages185-96
AuthorsJarret C, Stauffer F, Henz ME, Marty M, Luond RM, Bobalova J, Schurmann P, Neier R
TitleInhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.
[28]
CommentsX-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS).
Medline ID21338255
PubMed ID11444968
JournalBiochemistry
Year2001
Volume40
Pages8227-36
AuthorsKervinen J, Jaffe EK, Stauffer F, Neier R, Wlodawer A, Zdanov A
TitleMechanistic basis for suicide inactivation of porphobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity.
Related PDB1i8j
Related UniProtKBP15002
[29]
CommentsX-RAY CRYSTALLOGRAPHY (1.75 ANGSTROMS).
Medline ID21405508
PubMed ID11513881
JournalFEBS Lett
Year2001
Volume503
Pages196-200
AuthorsErskine PT, Coates L, Newbold R, Brindley AA, Stauffer F, Wood SP, Warren MJ, Cooper JB, Shoolingin-Jordan PM, Neier R
TitleThe X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors.
Related PDB1eb3,1gjp
Related UniProtKBP05373
[30]
PubMed ID11032841
JournalJ Biol Chem
Year2001
Volume276
Pages1538-44
AuthorsMitchell LW, Volin M, Martins J, Jaffe EK
TitleMechanistic implications of mutations to the active site lysine of porphobilinogen synthase.
[31]
CommentsX-RAY CRYSTALLOGRAPHY (1.6 ANGSTROMS).
Medline ID21431818
PubMed ID11545591
JournalJ Mol Biol
Year2001
Volume312
Pages133-41
AuthorsErskine PT, Newbold R, Brindley AA, Wood SP, Shoolingin-Jordan PM, Warren MJ, Cooper JB
TitleThe x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.
Related PDB1h7n,1h7o,1h7p,1h7r
Related UniProtKBP05373
[32]
PubMed ID11275419
JournalToxicol Lett
Year2001
Volume119
Pages27-37
AuthorsFarina M, Folmer V, Bolzan RC, Andrade LH, Zeni G, Braga AL, Rocha JB
TitleSelenoxides inhibit delta-aminolevulinic acid dehydratase.
[33]
PubMed ID12196142
JournalBiochem Soc Trans
Year2002
Volume30
Pages584-90
AuthorsShoolingin-Jordan PM, Spencer P, Sarwar M, Erskine PE, Cheung KM, Cooper JB, Norton EB
Title5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism.
[34]
CommentsX-RAY CRYSTALLOGRAPHY (1.7 ANGSTROMS).
Medline ID22028088
PubMed ID11909869
JournalJ Biol Chem
Year2002
Volume277
Pages19792-9
AuthorsJaffe EK, Kervinen J, Martins J, Stauffer F, Neier R, Wlodawer A, Zdanov A
TitleSpecies-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid.
Related PDB1l6s,1l6y
Related UniProtKBP15002
[35]
PubMed ID12079382
JournalJ Mol Biol
Year2002
Volume320
Pages237-47
AuthorsFrere F, Schubert WD, Stauffer F, Frankenberg N, Neier R, Jahn D, Heinz DW
TitleStructure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism.
Related PDB1gzg
[36]
CommentsX-RAY CRYSTALLOGRAPHY (1.66 ANGSTROMS) OF MUTANT D139N.
Medline ID22075365
PubMed ID12777167
JournalBiochem J
Year2003
Volume373
Pages733-8
AuthorsErskine PT, Coates L, Butler D, Youell JH, Brindley AA, Wood SP, Warren MJ, Shoolingin-Jordan PM, Cooper JB
TitleX-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase.
Related PDB1ohl
Related UniProtKBQ59643
[37]
PubMed ID12897770
JournalNat Struct Biol
Year2003
Volume10
Pages757-63
AuthorsBreinig S, Kervinen J, Stith L, Wasson AS, Fairman R, Wlodawer A, Zdanov A, Jaffe EK
TitleControl of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.
Related PDB1pv8
[38]
PubMed ID15327955
JournalJ Mol Biol
Year2004
Volume342
Pages563-70
AuthorsCoates L, Beaven G, Erskine PT, Beale SI, Avissar YJ, Gill R, Mohammed F, Wood SP, Shoolingin-Jordan P, Cooper JB
TitleThe X-ray structure of the plant like 5-aminolaevulinic acid dehydratase from Chlorobium vibrioforme complexed with the inhibitor laevulinic acid at 2.6 A resolution.
Related PDB1w1z
[39]
PubMed ID15644204
JournalJ Mol Biol
Year2005
Volume345
Pages1059-70
AuthorsFrere F, Reents H, Schubert WD, Heinz DW, Jahn D
TitleTracking the evolution of porphobilinogen synthase metal dependence in vitro.
Related PDB1w54,1w56,1w5m,1w5n,1w5o,1w5p,1w5q
[40]
PubMed ID16131755
JournalActa Crystallogr D Biol Crystallogr
Year2005
Volume61
Pages1222-6
AuthorsErskine PT, Coates L, Newbold R, Brindley AA, Stauffer F, Beaven GD, Gill R, Coker A, Wood SP, Warren MJ, Shoolingin-Jordan PM, Neier R, Cooper JB
TitleStructure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid.
Related PDB2c1h
[41]
PubMed ID16304458
JournalActa Crystallogr D Biol Crystallogr
Year2005
Volume61
Pages1594-8
AuthorsCoates L, Beaven G, Erskine PT, Beale SI, Wood SP, Shoolingin-Jordan PM, Cooper JB
TitleStructure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase complexed with a diacid inhibitor.
Related PDB1w31
[42]
PubMed ID16819823
JournalBiochemistry
Year2006
Volume45
Pages8243-53
AuthorsFrere F, Nentwich M, Gacond S, Heinz DW, Neier R, Frankenberg-Dinkel N
TitleProbing the active site of Pseudomonas aeruginosa porphobilinogen synthase using newly developed inhibitors.
Related PDB2c13,2c14,2c15,2c16,2c18,2c19

comments
There are several subfamilies of the homologous enzymes, such as zinc-dependent (S00239, S00538 in EzCatDB), and zinc-independent (S00537, S00539) subfamilies. This enzyme belongs to zinc-dependent subfamily.
According to the literature [36], the reaction proceeds as follows:
(A) Exchange of double-bonded atoms; Schiff-base formation between Lys246 and P-side ALA substrate:
(B) Exchange of double-bonded atoms; Schiff-base formation between Lys194 and A-side ALA substrate:
(C) Isomerization; Shift of double-bond from N=C-C to N-C=C, forming an enamine intermediate at A-side:
(D) Addition (C-C bond formation):
(E) Addition (C-N bond formation):
(F) Eliminative double-bond formation (releasing Lys194 at A-side):
(G) Eliminative double-bond formation (releasing Lys246 at P-side):
(H) Isomerization; Shift of double-bonds from N=C-C=C-C to N-C=C-C=C:
However, although the first two reactions (A-B) and the last reaction (H) seem to be common to all the homologous enzymes (S00537, S00538, S00539 in EzCatDB), the rest of the reactions (D-G) have not been elucidated yet.
The detailed mechanism must be as follows:
(A) Exchange of double-bonded atoms; Schiff-base formation between Lys246 and P-side ALA substrate (see [17], [27] and [31]):
(A1) Lys194 acts as a modulator to lower the pKa of the nucleophile Lys246.
(A2) Lys246 makes a nucleophilic attack on the carbonyl carbon of the substrate, ALA, at P-side, leading to formation of a tetrahedral carbinolamine transition-state. The carbinolamine oxygen of the transition-state is stabilized by the positive charge of Lys194.
(A3) Lys194 acts as a general acid to protonate the carbinolamine oxygen of the transition-state, leading to formation of hydroxyl group, and then acts as a general base to deprotonate the sidechain of Lys246.
(A4) The lone pair of the amine nitrogen of Lys246 makes a nucleophilic attack on the carbon atom of the carbinolamine transition-state, forming a Schiff-base. At the same time, Lys194 acts as a general acid to protonate the hydroxyl group of the carbinolamine, to facilitate the elimination of water molecule.
(B) Exchange of double-bonded atoms; Schiff-base formation between Lys194 and A-side ALA substrate:
(B1) Unprotonated sidechain of Lys194 acts as a nucleophile to attack on the carbonyl carbon of the substrate, ALA, at A-side, leading to formation of a carbinolamine transition-state. The carbinolamine oxygen of the transition-state is stabilized by the positive charge of the amino group of the ALA substrate at A-side.
(B2) The amino group of the ALA substrate at A-side might act as a general acid to protonate the carbinolamine oxygen of the transition-state, leading to formation of hydroxyl group, and then act as a general base to deprotonate the sidechain of Lys194.
(B3) The lone pair of the amine nitrogen of Lys194 makes a nucleophilic attack on the carbon atom of the carbinolamine transition-state, forming a Schiff-base. At the same time, the amino group of the ALA acts as a general acid to protonate the hydroxyl group of the carbinolamine, to facilitate the elimination of water molecule.
(C) Isomerization; Shift of double-bond from N=C-C to N-C=C, forming an enamine intermediate at A-side:
(C1) Hydroxide bound to zinc ion acts as a strong base to deprotonate C3 methylene of the Schiff-base intermediate at A-side, leading to formation of an enamine intermediate.
(E) Addition (C-N bond formation):
(E1) The unprotonated amino group of the ALA at A-side acts as a general base to deprotonate the amino group of the ALA at P-side.
(E2) The amino group of the ALA at P-side makes a nucleophilic attack on the Schiff base of A-side, forming a cyclic intermediate.
(E3) The protonated amino group of the ALA at A-side acts as a general acid to protonate the sidechain amine group of Lys194 at A-side.
(F) Eliminative double-bond formation (releasing Lys194 at A-side):
(F1) Lys246, which is covalently bound to the cyclic intermediate, acts as a general base to deprotonate the C2 atom of the intermediate (or originally C3 atom of ALA at A-side), and then acts as a general acid to protonate the eliminated group, Lys210.
(F2) At the same time, Lys210 is eliminated from the intermediate; E2-like reaction.
(G) Eliminative double-bond formation (releasing Lys246 at P-side):
(G1) Lys210 acts as a general acid to protonate the eliminated group, the amino group of Lys246.
(G2) The lone pair of enamine of the intermediate push the electron to form a new double bond, facilitating the elimination of Lys246; E1-like reaction.
(H) Isomerization; Shift of double-bonds from N=C-C=C-C to N-C=C-C=C:
(H1) Electrophilic Schiff-base assisted reaction. Lys246 acts as a general base to the C4 methylene of the intermediate (or originally C5 atom of ALA at P-side), leading to the shift of double-bonds.

createdupdated
2004-06-222010-05-19


Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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