DB code: D00273

RLCP classification	8.131.581400.399 : Isomerization
RLCP classification	8.113.580000.386 : Isomerization
CATH domain	3.30.390.10 : Enolase-like; domain 1
CATH domain	3.20.20.120 : TIM Barrel	Catalytic domain
E.C.	5.1.2.2
CSA	1mdr
M-CSA	1mdr
MACiE	M0187

CATH domain	Related DB codes (homologues)
3.20.20.120 : TIM Barrel	D00261 D00282 D00283
3.30.390.10 : Enolase-like; domain 1	D00261 D00282 D00283

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P11444	Mandelate racemase	MR EC 5.1.2.2	PF01188 (MR_MLE) PF02746 (MR_MLE_N) [Graphical View]

KEGG enzyme name
mandelate racemase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P11444	MANR_PSEPU	(S)-mandelate = (R)-mandelate.	Homooctamer.		Divalent metal ions. Magnesium seems to be the preferred ion.

KEGG Pathways
Map code	Pathways	E.C.
MAP00362	Benzoate degradation via hydroxylation
MAP00622	Toluene and xylene degradation

Compound table
	Cofactors	Substrates	Products	Intermediates
KEGG-id	C00305	C01984	C01983	I00074
E.C.
Compound	Magnesium	(S)-Mandelate	(R)-Mandelate	Phenylethene-1,2-triol
Type	divalent metal (Ca2+, Mg2+)	aromatic ring (only carbon atom),carbohydrate,carboxyl group	aromatic ring (only carbon atom),carbohydrate,carboxyl group
ChEBI	18420 18420	32800 32800	17656 17656
PubChem	888 888	439616 439616	11914 11914

1dtnA01	Unbound	Unbound	Unbound	Unbound
1mdlA01	Unbound	Unbound	Unbound	Unbound
1mdrA01	Unbound	Unbound	Unbound	Unbound
1mnsA01	Unbound	Unbound	Unbound	Unbound
1mraA01	Unbound	Unbound	Unbound	Unbound
2mnrA01	Unbound	Unbound	Unbound	Unbound
1dtnA02	Bound:_MG	Analogue:APG	Unbound	Unbound
1mdlA02	Bound:_MG	Bound:SMN	Unbound	Unbound
1mdrA02	Bound:_MG	Analogue:APG	Unbound	Unbound
1mnsA02	Bound:_MG	Analogue:APG	Unbound	Unbound
1mraA02	Bound:_MG	Analogue:APG	Unbound	Unbound
2mnrA02	Analogue:_MN	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
Swiss-prot;P11444 & literature [4], [9], [10], [11], [12], [14]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1dtnA01
1mdlA01
1mdrA01
1mnsA01
1mraA01
2mnrA01
1dtnA02	LYS 164;LYS 166;ASP 270;HIS 297;	ASP 195;GLU 221;GLU 247(Magnesium binding)			mutant E317Q
1mdlA02	LYS 164;;ASP 270;HIS 297;GLU 317	ASP 195;GLU 221;GLU 247(Magnesium binding)			mutant K166R
1mdrA02	LYS 164;LYS 166;ASP 270;HIS 297;GLU 317	ASP 195;GLU 221;GLU 247(Magnesium binding)
1mnsA02	LYS 164;LYS 166;ASP 270;HIS 297;GLU 317	ASP 195;GLU 221;GLU 247(Magnesium binding)	LYS 166(APG binding)
1mraA02	LYS 164;LYS 166;;HIS 297;GLU 317	ASP 195;GLU 221;GLU 247(Magnesium binding)			mutant D270N
2mnrA02	LYS 164;LYS 166;ASP 270;HIS 297;GLU 317	ASP 195;GLU 221;GLU 247(Magnesium binding)

References for Catalytic Mechanism
References	Sections	No. 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]
Resource
Comments
Medline ID
PubMed ID	3132459
Journal	J Biol Chem
Year	1988
Volume	263
Pages	9268-70
Authors	Neidhart DJ, Powers VM, Kenyon GL, Tsou AY, Ransom SC, Gerlt JA, Petsko GA
Title	Preliminary x-ray data on crystals of mandelate racemase.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID	2099737
Journal	Biochem Soc Symp
Year	1990
Volume	57
Pages	135-41
Authors	Neidhart DC, Howell PL, Petsko GA, Gerlt JA, Kozarich JW, Powers VM, Kenyon GL
Title	Restructuring catalysis in the mandelate pathway.
Related PDB
Related UniProtKB
[3]
Resource
Comments	SIMILARITY TO MLE.
Medline ID	91015392
PubMed ID	2215699
Journal	Nature
Year	1990
Volume	347
Pages	692-4
Authors	Neidhart DJ, Kenyon GL, Gerlt JA, Petsko GA
Title	Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous.
Related PDB
Related UniProtKB	P11444
[4]
Resource
Comments	MUTAGENESIS OF HIS-297.
Medline ID	91369941
PubMed ID	1909893
Journal	Biochemistry
Year	1991
Volume	30
Pages	9274-81
Authors	Landro JA, Kallarakal AT, Ransom SC, Gerlt JA, Kozarich JW, Neidhart DJ, Kenyon GL
Title	Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant.
Related PDB
Related UniProtKB	P11444
[5]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS).
Medline ID	91369940
PubMed ID	1892834
Journal	Biochemistry
Year	1991
Volume	30
Pages	9264-73
Authors	Neidhart DJ, Howell PL, Petsko GA, Powers VM, Li RS, Kenyon GL, Gerlt JA
Title	Mechanism 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 PDB	2mnr
Related UniProtKB	P11444
[6]
Resource
Comments
Medline ID
PubMed ID	1892833
Journal	Biochemistry
Year	1991
Volume	30
Pages	9255-63
Authors	Powers VM, Koo CW, Kenyon GL, Gerlt JA, Kozarich JW
Title	Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	1986411
Journal	Science
Year	1991
Volume	251
Pages	31-2
Authors	Hoffman M
Title	On the road to mandelate ... racemase.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	8256284
Journal	Trends Biochem Sci
Year	1993
Volume	18
Pages	372-6
Authors	Petsko GA, Kenyon GL, Gerlt JA, Ringe D, Kozarich JW
Title	On the origin of enzymatic species.
Related PDB
Related UniProtKB
[9]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	8292591
Journal	Biochemistry
Year	1994
Volume	33
Pages	635-43
Authors	Landro JA, Gerlt JA, Kozarich JW, Koo CW, Shah VJ, Kenyon GL, Neidhart DJ, Fujita S, Petsko GA
Title	The 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 PDB	1mdr 1mns
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID	8009219
Journal	Science
Year	1994
Volume	264
Pages	1887-90
Authors	Cleland WW, Kreevoy MM
Title	Low-barrier hydrogen bonds and enzymic catalysis.
Related PDB
Related UniProtKB
[11]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.85 ANGSTROMS).
Medline ID	95200898
PubMed ID	7893690
Journal	Biochemistry
Year	1995
Volume	34
Pages	2788-97
Authors	Kallarakal AT, Mitra B, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
Title	Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the K166R mutant.
Related PDB	1mdl
Related UniProtKB	P11444
[12]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS).
Medline ID	95200897
PubMed ID	7893689
Journal	Biochemistry
Year	1995
Volume	34
Pages	2777-87
Authors	Mitra B, Kallarakal AT, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
Title	Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.
Related PDB	1dtn
Related UniProtKB	P11444
[13]
Resource
Comments
Medline ID
PubMed ID	8987982
Journal	Biochemistry
Year	1996
Volume	35
Pages	16489-501
Authors	Babbitt PC, Hasson MS, Wedekind JE, Palmer DR, Barrett WC, Reed GH, Rayment I, Ringe D, Kenyon GL, Gerlt JA
Title	The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.
Related PDB
Related UniProtKB
[14]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	8639525
Journal	Biochemistry
Year	1996
Volume	35
Pages	5662-9
Authors	Schafer SL, Barrett WC, Kallarakal AT, Mitra B, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL
Title	Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N mutant.
Related PDB	1mra
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID	9048548
Journal	Biochemistry
Year	1997
Volume	36
Pages	1646-56
Authors	Bearne SL, Wolfenden R
Title	Mandelate racemase in pieces: effective concentrations of enzyme functional groups in the transition state.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID	9772161
Journal	Biochemistry
Year	1998
Volume	37
Pages	14358-68
Authors	Gulick AM, Palmer DR, Babbitt PC, Gerlt JA, Rayment I
Title	Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID	9748211
Journal	J Biol Chem
Year	1998
Volume	273
Pages	25529-32
Authors	Cleland WW, Frey PA, Gerlt JA
Title	The low barrier hydrogen bond in enzymatic catalysis.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID	9724714
Journal	Proc Natl Acad Sci U S A
Year	1998
Volume	95
Pages	10396-401
Authors	Hasson MS, Schlichting I, Moulai J, Taylor K, Barrett W, Kenyon GL, Babbitt PC, Gerlt JA, Petsko GA, Ringe D
Title	Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID	11063568
Journal	Biochemistry
Year	2000
Volume	39
Pages	13324-35
Authors	St Maurice M, Bearne SL
Title	Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis.
Related PDB
Related UniProtKB
[20]
Resource
Comments
Medline ID
PubMed ID	11900548
Journal	Biochemistry
Year	2002
Volume	41
Pages	4048-58
Authors	St Maurice M, Bearne SL
Title	Kinetics and thermodynamics of mandelate racemase catalysis.
Related PDB
Related UniProtKB
[21]
Resource
Comments
Medline ID
PubMed ID	12781191
Journal	Bioorg Med Chem Lett
Year	2003
Volume	13
Pages	2041-4
Authors	St Maurice M, Bearne SL, Lu W, Taylor SD
Title	Inhibition of mandelate racemase by alpha-fluorobenzylphosphonates.
Related PDB
Related UniProtKB
[22]
Resource
Comments
Medline ID
PubMed ID	15023082
Journal	Acc Chem Res
Year	2004
Volume	37
Pages	149-58
Authors	Wise EL, Rayment I
Title	Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes.
Related PDB
Related UniProtKB
[23]
Resource
Comments
Medline ID
PubMed ID	14992589
Journal	Biochemistry
Year	2004
Volume	43
Pages	2524-32
Authors	St Maurice M, Bearne SL
Title	Hydrophobic nature of the active site of mandelate racemase.
Related PDB
Related UniProtKB
[24]
Resource
Comments
Medline ID
PubMed ID	15697231
Journal	Biochemistry
Year	2005
Volume	44
Pages	2059-71
Authors	Kalyanaraman C, Bernacki K, Jacobson MP
Title	Virtual screening against highly charged active sites: identifying substrates of alpha-beta barrel enzymes.
Related PDB
Related UniProtKB

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.

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.

Created	Updated
2005-05-11	2010-08-06