EzCatDB: D00273

DB codeD00273
RLCP classification8.131.581400.399
8.113.580000.386
CATH domainDomain 13.30.390.10
Domain 23.20.20.120Catalytic domain
E.C.5.1.2.2
CSA1mdr
MACiEM0187

CATH domainRelated DB codes (homologues)
3.20.20.120D00261,D00282,D00283
3.30.390.10D00261,D00282,D00283

Enzyme Name
Swiss-protKEGG

P11444
Protein nameMandelate racemasemandelate racemase
SynonymsMR
EC 5.1.2.2

KEGG pathways
MAP codePathways
MAP00362Benzoate degradation via hydroxylation
MAP00622Toluene and xylene degradation

Swiss-prot:Accession NumberP11444
Entry nameMANR_PSEPU
Activity(S)-mandelate = (R)-mandelate.
SubunitHomooctamer.
Subcellular location
CofactorDivalent metal ions. Magnesium seems to be the preferred ion.


CofactorsSubstratesProductsintermediates
KEGG-idC00305C01984C01983I00074
CompoundMagnesium(S)-Mandelate(R)-MandelatePhenylethene-1,2-triol
Typedivalent metal (Ca2+, Mg2+)aromatic ring (only carbon atom),carbohydrate,carboxyl grouparomatic ring (only carbon atom),carbohydrate,carboxyl group
1dtnA01UnboundUnboundUnboundUnbound
1mdlA01UnboundUnboundUnboundUnbound
1mdrA01UnboundUnboundUnboundUnbound
1mnsA01UnboundUnboundUnboundUnbound
1mraA01UnboundUnboundUnboundUnbound
2mnrA01UnboundUnboundUnboundUnbound
1dtnA02Bound:_MGAnalogue:APGUnboundUnbound
1mdlA02Bound:_MGBound:SMNUnboundUnbound
1mdrA02Bound:_MGAnalogue:APGUnboundUnbound
1mnsA02Bound:_MGAnalogue:APGUnboundUnbound
1mraA02Bound:_MGAnalogue:APGUnboundUnbound
2mnrA02Analogue:_MNUnboundUnboundUnbound

Active-site residues
resource
Swiss-prot;P11444 & literature [4], [9], [10], [11], [12], [14]
pdbCatalytic residuesCofactor-binding residuesModified residuescomment
1dtnA01



1mdlA01



1mdrA01



1mnsA01



1mraA01



2mnrA01



1dtnA02LYS 164;LYS 166;ASP 270;HIS 297;       
ASP 195;GLU 221;GLU 247(Magnesium binding)

mutant E317Q
1mdlA02LYS 164;       ;ASP 270;HIS 297;GLU 317
ASP 195;GLU 221;GLU 247(Magnesium binding)

mutant K166R
1mdrA02LYS 164;LYS 166;ASP 270;HIS 297;GLU 317
ASP 195;GLU 221;GLU 247(Magnesium binding)


1mnsA02LYS 164;LYS 166;ASP 270;HIS 297;GLU 317
ASP 195;GLU 221;GLU 247(Magnesium binding)
LYS 166(APG binding)

1mraA02LYS 164;LYS 166;       ;HIS 297;GLU 317
ASP 195;GLU 221;GLU 247(Magnesium binding)

mutant D270N
2mnrA02LYS 164;LYS 166;ASP 270;HIS 297;GLU 317
ASP 195;GLU 221;GLU 247(Magnesium binding)



References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[9]Scheme I
[10]Fig.6, p.1889
[11]p.2795-2796
[12]Fig.2, p.2782-2783
[13]p.16495-16496
[14]p.5665-5667
[15]Fig.7, p.1652-1654
[17]p.25532
[18]Fig.1, p.10400-10401
[19]Scheme 1, p.13332-13334
[22]p.152-153
[23]Scheme 1

references
[1]
PubMed ID3132459
JournalJ Biol Chem
Year1988
Volume263
Pages9268-70
AuthorsNeidhart DJ, Powers VM, Kenyon GL, Tsou AY, Ransom SC, Gerlt JA, Petsko GA
TitlePreliminary x-ray data on crystals of mandelate racemase.
[2]
PubMed ID2099737
JournalBiochem Soc Symp
Year1990
Volume57
Pages135-41
AuthorsNeidhart DC, Howell PL, Petsko GA, Gerlt JA, Kozarich JW, Powers VM, Kenyon GL
TitleRestructuring catalysis in the mandelate pathway.
[3]
CommentsSIMILARITY TO MLE.
Medline ID91015392
PubMed ID2215699
JournalNature
Year1990
Volume347
Pages692-4
AuthorsNeidhart DJ, Kenyon GL, Gerlt JA, Petsko GA
TitleMandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous.
Related Swiss-protP11444
[4]
CommentsMUTAGENESIS OF HIS-297.
Medline ID91369941
PubMed ID1909893
JournalBiochemistry
Year1991
Volume30
Pages9274-81
AuthorsLandro JA, Kallarakal AT, Ransom SC, Gerlt JA, Kozarich JW, Neidhart DJ, Kenyon GL
TitleMechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant.
Related Swiss-protP11444
[5]
CommentsX-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS).
Medline ID91369940
PubMed ID1892834
JournalBiochemistry
Year1991
Volume30
Pages9264-73
AuthorsNeidhart DJ, Howell PL, Petsko GA, Powers VM, Li RS, Kenyon GL, Gerlt JA
TitleMechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-A resolution: identification of the active site and possible catalytic residues.
Related PDB2mnr
Related Swiss-protP11444
[6]
PubMed ID1892833
JournalBiochemistry
Year1991
Volume30
Pages9255-63
AuthorsPowers VM, Koo CW, Kenyon GL, Gerlt JA, Kozarich JW
TitleMechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism.
[7]
PubMed ID1986411
JournalScience
Year1991
Volume251
Pages31-2
AuthorsHoffman M
TitleOn the road to mandelate . racemase.
[8]
PubMed ID8256284
JournalTrends Biochem Sci
Year1993
Volume18
Pages372-6
AuthorsPetsko GA, Kenyon GL, Gerlt JA, Ringe D, Kozarich JW
TitleOn the origin of enzymatic species.
[9]
CommentsX-ray crystallography
PubMed ID8292591
JournalBiochemistry
Year1994
Volume33
Pages635-43
AuthorsLandro JA, Gerlt JA, Kozarich JW, Koo CW, Shah VJ, Kenyon GL, Neidhart DJ, Fujita S, Petsko GA
TitleThe role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate.
Related PDB1mdr,1mns
[10]
PubMed ID8009219
JournalScience
Year1994
Volume264
Pages1887-90
AuthorsCleland WW, Kreevoy MM
TitleLow-barrier hydrogen bonds and enzymic catalysis.
[11]
CommentsX-RAY CRYSTALLOGRAPHY (1.85 ANGSTROMS).
Medline ID95200898
PubMed ID7893690
JournalBiochemistry
Year1995
Volume34
Pages2788-97
AuthorsKallarakal AT, Mitra B, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
TitleMechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the K166R mutant.
Related PDB1mdl
Related Swiss-protP11444
[12]
CommentsX-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS).
Medline ID95200897
PubMed ID7893689
JournalBiochemistry
Year1995
Volume34
Pages2777-87
AuthorsMitra B, Kallarakal AT, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
TitleMechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.
Related PDB1dtn
Related Swiss-protP11444
[13]
PubMed ID8987982
JournalBiochemistry
Year1996
Volume35
Pages16489-501
AuthorsBabbitt PC, Hasson MS, Wedekind JE, Palmer DR, Barrett WC, Reed GH, Rayment I, Ringe D, Kenyon GL, Gerlt JA
TitleThe enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.
[14]
CommentsX-ray crystallography
PubMed ID8639525
JournalBiochemistry
Year1996
Volume35
Pages5662-9
AuthorsSchafer SL, Barrett WC, Kallarakal AT, Mitra B, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
TitleMechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N mutant.
Related PDB1mra
[15]
PubMed ID9048548
JournalBiochemistry
Year1997
Volume36
Pages1646-56
AuthorsBearne SL, Wolfenden R
TitleMandelate racemase in pieces: effective concentrations of enzyme functional groups in the transition state.
[16]
PubMed ID9772161
JournalBiochemistry
Year1998
Volume37
Pages14358-68
AuthorsGulick AM, Palmer DR, Babbitt PC, Gerlt JA, Rayment I
TitleEvolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.
[17]
PubMed ID9748211
JournalJ Biol Chem
Year1998
Volume273
Pages25529-32
AuthorsCleland WW, Frey PA, Gerlt JA
TitleThe low barrier hydrogen bond in enzymatic catalysis.
[18]
PubMed ID9724714
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages10396-401
AuthorsHasson MS, Schlichting I, Moulai J, Taylor K, Barrett W, Kenyon GL, Babbitt PC, Gerlt JA, Petsko GA, Ringe D
TitleEvolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase.
[19]
PubMed ID11063568
JournalBiochemistry
Year2000
Volume39
Pages13324-35
AuthorsSt Maurice M, Bearne SL
TitleReaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis.
[20]
PubMed ID11900548
JournalBiochemistry
Year2002
Volume41
Pages4048-58
AuthorsSt Maurice M, Bearne SL
TitleKinetics and thermodynamics of mandelate racemase catalysis.
[21]
PubMed ID12781191
JournalBioorg Med Chem Lett
Year2003
Volume13
Pages2041-4
AuthorsSt Maurice M, Bearne SL, Lu W, Taylor SD
TitleInhibition of mandelate racemase by alpha-fluorobenzylphosphonates.
[22]
PubMed ID15023082
JournalAcc Chem Res
Year2004
Volume37
Pages149-58
AuthorsWise EL, Rayment I
TitleUnderstanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes.
[23]
PubMed ID14992589
JournalBiochemistry
Year2004
Volume43
Pages2524-32
AuthorsSt Maurice M, Bearne SL
TitleHydrophobic nature of the active site of mandelate racemase.
[24]
PubMed ID15697231
JournalBiochemistry
Year2005
Volume44
Pages2059-71
AuthorsKalyanaraman C, Bernacki K, Jacobson MP
TitleVirtual screening against highly charged active sites: identifying substrates of alpha-beta barrel enzymes.

comments
This enzyme catalyzes the following reactions, to produce (R)-mandelate from (S)-mandelate:
(A) Isomerization; Shift of double-bond position (from O=C-C to O-C=C), forming an enolic intermediate.
(B) Isomerization; Shift of double-bond position (from C=C-O to C-C=O).
According to the literature [11], [12], [14], [18] & [22], the catalytic reaction proceeds as follows:
(A) Isomerization; Shift of double-bond position (from O=C-C to O-C=C), forming an enolic intermediate.
(A1) A carboxylate oxygen and hydroxyl oxygen are coordinated to the cofactor, magnesium ion. (This ion must neutralize the negative charge on the carboxylate oxygen atom, along with Lys164.)
(A2) Lys166 acts as (S)-specific base to deprotonate the alpha-proton (from the single-bonded carbon) of the substrate, whereas glu317 acts as a general acid to protonate the carboxylate (double-bonded) oxygen, forming an enolic intermediate.
(A3) Here, Glu317 must stabilize the intermediate, by neutralizing the negative charge on the oxygen atom.
(B) Isomerization; Shift of double-bond position (from C=C-O to C-C=O).
(B1) The enol oxygen and hydroxyl oxygen are coordinated to the cofactor, magnesium ion. (This ion must neutralize the negative charge on the enol oxygen atom, along with Lys164.) Glu317 also stabilizes the other enol oxygen.
(B2) Asp270 modulates the activity of His297, as a pKa modulator.
(B3) His297 acts as (R)-specific acid to protonate the (double-bonded) carbon atom of the enol intermediate, whereas Glu317 acts as a general base to deprotonate the enol (single-bonded) oxygen. These completes the reaction.

createdupdated
2005-05-112010-08-06
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 2010)
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)

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