DB code: D00105

RLCP classification	6.30.97710.5300 : Double-bonded atom exchange
	8.211.591510.5526 : Isomerization
	6.20.85210.5511 : Double-bonded atom exchange
	6.10.85210.5901 : Double-bonded atom exchange
	8.211.591510.5527 : Isomerization
	6.40.521010.5520 : Double-bonded atom exchange
CATH domain	3.30.470.10 : D-amino Acid Aminotransferase; Chain A, domain 1	Catalytic domain
CATH domain	3.20.10.10 : D-amino Acid Aminotransferase; Chain A, domain 2	Catalytic domain
E.C.	2.6.1.21
CSA	1daa
M-CSA	1daa
MACiE	M0066

CATH domain	Related DB codes (homologues)
3.20.10.10 : D-amino Acid Aminotransferase; Chain A, domain 2	D00106
3.30.470.10 : D-amino Acid Aminotransferase; Chain A, domain 1	D00106

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P19938	D-alanine aminotransferase	EC 2.6.1.21 D-amino acid aminotransferase D-amino acid transaminase DAAT D-aspartate aminotransferase	PF01063 (Aminotran_4) [Graphical View]

KEGG enzyme name
D-amino-acid transaminase D-aspartate transaminase D-alanine aminotransferase D-aspartic aminotransferase D-alanine-D-glutamate transaminase D-alanine transaminase D-amino acid aminotransferase

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P19938	DAAA_BACYM	D-alanine + 2-oxoglutarate = pyruvate + D- glutamate.	Homodimer.		Pyridoxal phosphate.

KEGG Pathways
Map code	Pathways	E.C.
MAP00310	Lysine degradation
MAP00330	Arginine and proline metabolism
MAP00360	Phenylalanine metabolism
MAP00472	D-Arginine and D-ornithine metabolism
MAP00473	D-Alanine metabolism
MAP00550	Peptidoglycan biosynthesis

Compound table
	Cofactors	Substrates		Products		Intermediates
KEGG-id	C00018	C00133	C00026	C00022	C00217	I00029	I00032	I00030		C00647	I00006	I00033	I00031
E.C.									(carbinolabine)
Compound	Pyridoxal phosphate	D-Alanine	2-Oxoglutarate	Pyruvate	D-Glutamate	External aldimine intermediate (initial stage:PLP-D-Ala)	Quinonoid Intermediate-1 (PLP-Ala)	Ketimine intermediate-1 (PLP-Ala)	Tetrahedral intermediate from ketimine to PMP	Pyridoxamine phosphate (PMP)	Ketimine intermediate-2 (PLP-Glu)	Quinonoid Intermediate-2 (PLP-Glu)	External aldimine intermediate (final stage:PLP-D-Glu)
Type	aromatic ring (with nitrogen atoms),phosphate group/phosphate ion	amino acids	carbohydrate,carboxyl group	carbohydrate,carboxyl group	amino acids,carboxyl group
ChEBI	18405 18405	15570 57416 15570 57416	30915 30915	32816 32816	15966 15966
PubChem	1051 1051	71080 7311725 71080 7311725	51 51	1060 1060	23327 23327

1a0gA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1a0gB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1daaA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1daaB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1g2wA01	Unbound	Unbound	Unbound	Analogue:ACT	Unbound	Unbound	Unbound			Unbound
1g2wB01	Unbound	Unbound	Unbound	Analogue:ACT	Unbound	Unbound	Unbound			Unbound
2daaA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
2daaB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
2dabA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
2dabB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
3daaA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
3daaB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
4daaA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
4daaB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
5daaA01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
5daaB01	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1a0gA02	Analogue:PMP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Intermediate-bound:PMP
1a0gB02	Analogue:PMP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Intermediate-bound:PMP
1daaA02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1daaB02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1g2wA02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
1g2wB02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
2daaA02	Analogue:DCS	Unbound	Unbound	Unbound	Unbound	Unbound	Intermediate-analogue:DCS			Unbound
2daaB02	Analogue:DCS	Unbound	Unbound	Unbound	Unbound	Unbound	Intermediate-analogue:DCS			Unbound
2dabA02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
2dabB02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
3daaA02	Analogue:PDD	Unbound	Unbound	Unbound	Unbound	Intermediate-bound:PDD	Unbound			Unbound
3daaB02	Analogue:PDD	Unbound	Unbound	Unbound	Unbound	Intermediate-bound:PDD	Unbound			Unbound
4daaA02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
4daaB02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
5daaA02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound
5daaB02	Bound:PLP	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound			Unbound

Reference for Active-site residues
resource	references	E.C.
literature [8], [16], [19], [21]

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

1a0gA01	TYR 31;HIS 100
1a0gB01	TYR 31;HIS 100
1daaA01	TYR 31;HIS 100
1daaB01	TYR 31;HIS 100
1g2wA01	TYR 31;HIS 100
1g2wB01	TYR 31;HIS 100
2daaA01	TYR 31;HIS 100
2daaB01	TYR 31;HIS 100
2dabA01	TYR 31;HIS 100
2dabB01	TYR 31;HIS 100
3daaA01	TYR 31;HIS 100
3daaB01	TYR 31;HIS 100
4daaA01	TYR 31;HIS 100
4daaB01	TYR 31;HIS 100
5daaA01	TYR 31;HIS 100
5daaB01	TYR 31;HIS 100
1a0gA02	LYS 145;GLU 177	LYS 145(PLP binding)			mutant L201A
1a0gB02	LYS 145;GLU 177	LYS 145(PLP binding)			mutant L201A
1daaA02	LYS 145;GLU 177	LYS 145(PLP binding)
1daaB02	LYS 145;GLU 177	LYS 145(PLP binding)
1g2wA02	LYS 145;	LYS 145(PLP binding)			mutant E177S
1g2wB02	;				mutant E177S, invisible 145
2daaA02	LYS 145;GLU 177	LYS 145(PLP binding)
2daaB02	LYS 145;GLU 177	LYS 145(PLP binding)
2dabA02	LYS 145;GLU 177	LYS 145(PLP binding)			mutant L201A
2dabB02	LYS 145;GLU 177	LYS 145(PLP binding)			mutant L201A
3daaA02	LYS 145;GLU 177	LYS 145(PLP binding)
3daaB02	LYS 145;GLU 177	LYS 145(PLP binding)
4daaA02	LYS 145;GLU 177	LYS 145(PLP binding)
4daaB02	LYS 145;GLU 177	LYS 145(PLP binding)
5daaA02	LYS 145;	LYS 145(PLP binding)			mutant E177K
5daaB02	LYS 145;	LYS 145(PLP binding)			mutant E177K

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[8]	Scheme I
[9]	Fig.1, p.9661-9662, p.9666-9667
[13]	Fig.1, Fig.2, p.181-182
[14]	Fig.1
[16]	Fig.1, Fig.8, p.4964
[17]	Fig.1, p.765
[19]	Fig.1, p.617-618
[20]	Fig.1, Fig.3, p.696-697, p.698-699
[21]	p.1330-1331
[22]	Fig.3, Fig.10, p.143-149, p.155-156
[25]	Fig.2, p.R5
[26]	Fig.1, Fig.2, Fig.3, p.375-378
[27]	Fig.1
[28]	Fig.1

References
[1]
Resource
Comments
Medline ID
PubMed ID	4950474
Journal	Adv Enzymol Relat Areas Mol Biol
Year	1971
Volume	35
Pages	79-134
Authors	Dunathan HC
Title	Stereochemical aspects of pyridoxal phosphate catalysis.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID	2713327
Journal	Biochemistry
Year	1989
Volume	28
Pages	510-6
Authors	Martinez del Pozo A, Merola M, Ueno H, Manning JM, Tanizawa K, Nishimura K, Asano S, Tanaka H, Soda K, Ringe D, et al
Title	Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID	2496746
Journal	Biochemistry
Year	1989
Volume	28
Pages	505-9
Authors	Merola M, Martinez del Pozo A, Ueno H, Recsei P, Di Donato A, Manning JM, Tanizawa K, Masu Y, Asano S, Tanaka H, et al
Title	Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID	2914916
Journal	J Biol Chem
Year	1989
Volume	264
Pages	2445-9
Authors	Tanizawa K, Masu Y, Asano S, Tanaka H, Soda K
Title	Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	2125047
Journal	J Biol Chem
Year	1990
Volume	265
Pages	22306-12
Authors	Futaki S, Ueno H, Martinez del Pozo A, Pospischil MA, Manning JM, Ringe D, Stoddard B, Tanizawa K, Yoshimura T, Soda K
Title	Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	1902115
Journal	Biochemistry
Year	1991
Volume	30
Pages	4072-7
Authors	Nishimura K, Tanizawa K, Yoshimura T, Esaki N, Futaki S, Manning JM, Soda K
Title	Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	1445909
Journal	Biochemistry
Year	1992
Volume	31
Pages	11748-54
Authors	Yoshimura T, Bhatia MB, Manning JM, Ringe D, Soda K
Title	Partial reactions of bacterial D-amino acid transaminase with asparagine substituted for the lysine that binds coenzyme pyridoxal 5'-phosphate.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	8463224
Journal	J Biol Chem
Year	1993
Volume	268
Pages	6932-8
Authors	Bhatia MB, Futaki S, Ueno H, Manning JM, Ringe D, Yoshimura T, Soda K
Title	Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.
Related PDB
Related UniProtKB
[9]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (1.94 ANGSTROMS)
Medline ID	95352651
PubMed ID	7626635
Journal	Biochemistry
Year	1995
Volume	34
Pages	9661-9
Authors	Sugio S, Petsko GA, Manning JM, Soda K, Ringe D
Title	Crystal structure of a D-amino acid aminotransferase: how the protein controls stereoselectivity.
Related PDB	1daa
Related UniProtKB	P19938
[10]
Resource
Comments
Medline ID
PubMed ID	7592528
Journal	J Biochem (Tokyo)
Year	1995
Volume	117
Pages	691-6
Authors	Kishimoto K, Yoshimura T, Esaki N, Sugio S, Manning JM, Soda K
Title	Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID	8580849
Journal	Protein Sci
Year	1995
Volume	4
Pages	2578-86
Authors	Van Ophem PW, Pospischil MA, Ringe D, Peisach D, Petsko G, Soda K, Manning JM
Title	Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID	8652553
Journal	Biochemistry
Year	1996
Volume	35
Pages	2112-6
Authors	Martinez del Pozo A, van Ophem PW, Ringe D, Petsko G, Soda K, Manning JM
Title	Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID	9063963
Journal	Biosci Biotechnol Biochem
Year	1996
Volume	60
Pages	181-7
Authors	Yoshimura T, Jhee KH, Soda K
Title	Stereospecificity for the hydrogen transfer and molecular evolution of pyridoxal enzymes.
Related PDB
Related UniProtKB
[14]
Resource
Comments
Medline ID
PubMed ID	9498563
Journal	J Biochem (Tokyo)
Year	1997
Volume	122
Pages	1182-9
Authors	Kishimoto K, Yoshimura T, Soda K, Esaki N
Title	Mutation of arginine 98, which serves as a substrate-recognition site of D-amino acid aminotransferase, can be partly compensated for by mutation of tyrosine 88 to an arginyl residue.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID	9163511
Journal	J Biochem (Tokyo)
Year	1997
Volume	121
Pages	637-41
Authors	Okada K, Hirotsu K, Sato M, Hayashi H, Kagamiyama H
Title	Three-dimensional structure of Escherichia coli branched-chain amino acid aminotransferase at 2.5 A resolution.
Related PDB
Related UniProtKB
[16]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS)
Medline ID	98206914
PubMed ID	9538014
Journal	Biochemistry
Year	1998
Volume	37
Pages	4958-67
Authors	Peisach D, Chipman DM, Van Ophem PW, Manning JM, Ringe D
Title	Crystallographic study of steps along the reaction pathway of D-amino acid aminotransferase.
Related PDB	2daa 3daa 4daa
Related UniProtKB	P19938
[17]
Resource
Comments
Medline ID
PubMed ID	9914259
Journal	Curr Opin Struct Biol
Year	1998
Volume	8
Pages	759-69
Authors	Jansonius JN
Title	Structure, evolution and action of vitamin B6-dependent enzymes.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID	9792912
Journal	J Biochem (Tokyo)
Year	1998
Volume	124
Pages	905-10
Authors	Fuchikami Y, Yoshimura T, Gutierrez A, Soda K, Esaki N
Title	Construction and properties of a fragmentary D-amino acid aminotransferase.
Related PDB
Related UniProtKB
[19]
Resource
Comments	X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
Medline ID	98420361
PubMed ID	9749913
Journal	Protein Eng
Year	1998
Volume	11
Pages	613-9
Authors	Sugio S, Kashima A, Kishimoto K, Peisach D, Petsko GA, Ringe D, Yoshimura T, Esaki N
Title	Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 A resolution: implication of the structural role of Leu201 in transamination.
Related PDB	1a0g 2dab
Related UniProtKB	P19938
[20]
Resource
Comments
Medline ID
PubMed ID
Journal	J Microbiol Biotechnol
Year	1999
Volume	9
Pages	695-703
Authors	Jhee KH, Yoshimura T, Kurokawa Y, Esaki N, Soda K
Title	A stereochemical aspect of pyridoxal 5 '-phosphate dependent enzyme reactions and molecular evolution.
Related PDB
Related UniProtKB
[21]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	9930994
Journal	Biochemistry
Year	1999
Volume	38
Pages	1323-31
Authors	van Ophem PW, Peisach D, Erickson SD, Soda K, Ringe D, Manning JM
Title	Effects of the E177K mutation in D-amino acid transaminase. Studies on an essential coenzyme anchoring group that contributes to stereochemical fidelity.
Related PDB	5daa
Related UniProtKB
[22]
Resource
Comments
Medline ID
PubMed ID	10800595
Journal	Adv Enzymol Relat Areas Mol Biol
Year	2000
Volume	74
Pages	129-84
Authors	Mehta PK, Christen P
Title	The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.
Related PDB
Related UniProtKB
[23]
Resource
Comments
Medline ID
PubMed ID	10630999
Journal	Biochemistry
Year	2000
Volume	39
Pages	381-7
Authors	Kishimoto K, Yasuda C, Manning JM
Title	Reversible dissociation/association of D-amino acid transaminase subunits: properties of isolated active dimers and inactive monomers.
Related PDB
Related UniProtKB
[24]
Resource
Comments
Medline ID
PubMed ID	11106434
Journal	Eur J Biochem
Year	2000
Volume	267
Pages	7218-23
Authors	Gutierrez A, Yoshimura T, Fuchikami Y, Esaki N
Title	Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.
Related PDB
Related UniProtKB
[25]
Resource
Comments
Medline ID
PubMed ID	10673430
Journal	Structure Fold Des
Year	2000
Volume	8
Pages	R1-6
Authors	Schneider G, Kack H, Lindqvist Y
Title	The manifold of vitamin B6 dependent enzymes.
Related PDB
Related UniProtKB
[26]
Resource
Comments
Medline ID
PubMed ID	11933244
Journal	Chem Rec
Year	2001
Volume	1
Pages	373-84
Authors	Soda K, Yoshimura T, Esaki N
Title	Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions.
Related PDB
Related UniProtKB
[27]
Resource
Comments
Medline ID
PubMed ID	11642362
Journal	Prog Nucleic Acid Res Mol Biol
Year	2001
Volume	70
Pages	175-206
Authors	Hutson S
Title	Structure and function of branched chain aminotransferases.
Related PDB
Related UniProtKB
[28]
Resource
Comments
Medline ID
PubMed ID	12297014
Journal	J Biochem Mol Biol
Year	2002
Volume	35
Pages	306-12
Authors	Ro HS
Title	Effects of salts on the conformation and catalytic properties of d-amino acid aminotransferase.
Related PDB
Related UniProtKB

Comments
(B2) Lys145 acts as a general base to deprotonate the alpha-proton of the amino acid substrate, forming a quinonoid intermediate. (B3) Lys145 acts as a general acid to protonate the C4' atom of the PLP, leading to the formation of a ketimine intermediate. (C) Schiff-base deforming by hydration, releasing the first product, pyruvate, and PMP. (C0) Considering the active site of enzyme (PDB;2daa), a water molecule is unlikely to approach from the side of the active site residues, Tyr31 and Lys145. (C1) Thus, His100* from the adjacent subunit might act as a general base to activate the water molecule at the si-face side of cofactor. (C2) The activated water molecule makes a nucleophilic attack on the alpha-carbon atom of the substrate (from the si-face side), forming a carbinolamine intermediate. (C3) The protonated His100* might transfer the proton to Lys145, through a water molecule and either O3' atom of PLP or Tyr31. (C4) Lys145 may act as a general acid to protonate the N4' atom of the PLP. Tyr31 may modulate the activity of Lys145. (C5) The lone pair of the hydroxyl oxygen makes a nucleophilic attack on the C4' atom, whereas His100* acts as a general base to deprotonate the hydroxyl group. (C6) The protonated His100* might transfer the proton to Lys145, through a water molecule and either O3' atom of PLP or Tyr31. Finally, Lys145 act as a general acid to protonate the N4' atom, releasing the first product, pyruvate and the PMP. (D) Schiff-base forming of PMP with carbonyl group of the second substrate, 2-oxoglutarate. (D0) The second substrate, 2-oxoglutarate, is bound to the active site, with the carbonyl oxygen hydrogen-bonded by His100. (D1) Lys145 acts as a general base to deprotonate the amine group (or the N4' atom) of PMP. (D2) The deprotonated amine group (or the N4' atom) of PMP makes a nucleophilic attack on the carbonyl carbon of the substrate, whereas His100 acts as a general acid to protonate the carbonyl oxygen, forming a carbinolamine intermediate. (D3) The protonated Lys145 might transfer the proton to His100, through a water molecule and either O3' atom of PLP or Tyr31. (D4) Lys145 acts as a general base to deprotonate the N4' amine group. Tyr31 may modulate the activity of Lys258. (D5) The lone pair of the N4' nitrogen atom makes a nucleophilic attack on the carbon atom, whereas His100 acts as a general acid to the hydroxyl group of the carbinolamine intermediate, leading to the cleavage between the carbon and the N4' atom, and to the release of a water moleucle. This reaction gives a ketimine intermediate again. (E) Isomerization (change in the position of double-bond). (E1) Glu177 modulates and enhances the activity of the PLP cofactor as an electron sink, which facilitates the abstraction of alpha-proton from the C4' atom of the PLP. At the same time, Tyr31 seems to modulate the pKa of Lys145 sidechain, whereas the O3' atom of PLP interacts with the Schiff-base nitrogen atom, modulating the pKa (see [16]). (E2) Lys145 acts as a general base to deprotonate the C4' atom of the PLP, leading to the formation of a quinonoid intermediate. (E3) Lys145 acts as a general acid to protonate the alpha-proton of the amino acid substrate, forming an external aldimine. (As a result, Lys145 must be deprotonated, so that it can act as a general base at the next stage.) (F) Formation of internal aldimine, leading to the elimination of the product, D-glutamate, from PLP. (F1) Tyr31 might interact with O3' atom of PLP through a water , when the PLP-aldimine is protonated, modulating and keeping the O3' of PLP negatively charged (see [15], [16]). (F2) The negatively charged O3' atom of PLP modulates the pKa of the internal aldimine with Lys145 (see [16]). (F3) The deprotonated amine group of Lys145 makes a nucleophilic attack on the C4' carbon of the PLP of the external aldimine, forming a transient geminal diamine intermediate. (F4) There must be a general acid, which protonates the N4' nitrogen atom of the external aldimine or the geminal diamine. Considering the active-site structure, The adjacent Lys145 nitrogen may play the role as the general acid to protonate the N4' atom, through a water. This enzyme belongs to the fold-type IV pyridoxal-phosphate-dependent enzymes (see [17]). Unlike other PLP-dependent aminotransferases (D00101, D00102 in EzCatDB), the catalytic lysine (Lus145) of this enzyme transfer the pro-R hydrogen at the C4' position of pyridoxal phosphate (PLP) on the re face (see [9], [13]). However, the catalytic residues surrounding PLP of this enzyme is very similar to that of aspartate aminotransferase (D00101 in EzCatDB), according to the literature [15]. This enzyme catalyzes transamination, which is composed of the following reactions (see [16]): (A) Formation of external aldimine (with amine group of D-alanine). (B) Isomerization (change in the position of double-bond), forming a ketimine intermediate. (C) Schiff-base deforming by hydration, releasing the first product, pyruvate, and PMP. (D) Schiff-base forming of PMP with carbonyl group of the second substrate, 2-oxoglutarate, leaging again to a ketimine intermediate. (E) Isomerization (change in the position of double-bond). (F) Formation of internal aldimine, leading to the elimination of the product, D-glutamate, from PLP. These reactions proceed in the following way: (A) Formation of external aldimine (with amine group of D-alanine). (A1) Tyr31 might interact with O3' atom of PLP through a water together with the imine nitrogen of Lys145, when the PLP-aldimine is protonated, modulating and keeping the O3' of PLP negatively charged (see [15], [16]). (A2) The negatively charged O3' atom of PLP modulates the pKa of the internal aldimine with Lys145 (see [16]). (A3) The deprotonated amine group of D-alanine makes a nucleophilic attack on the C4' carbon of PLP, forming a transient geminal diamine intermediate (see [16]). (A4) There must be a general base, which deprotonates the amine group of the previously D-alanine substrate, so that the lone pair of the amine group can attack on the C4' atom to form a double-bond, and to release the amine of the catalytic residue, Lys145. The adjacent Lys145 nitrogen may act as a general base to deprotonate the amine group of the incoming substrate, through a water molecule. (see [16]) (A5) The reaction produces the external aldimine with D-alanine. (B) Isomerization (change in the position of double-bond), forming a ketimine intermediate. (B1) Glu177 interacts with the N1 atom of PLP, keeping the protonated state of the pyridinium nitrogen of PLP, so that it can modulate and enhance the activity of the PLP cofactor as an electron sink, which facilitates the abstraction of alpha-proton from the D-alanine covalently bound to the PLP (see [16] & [21]). At the same time, Tyr31 seems to modulate the pKa of Lys145 sidechain, whereas the O3' atom of PLP interacts with the Schiff-base nitrogen atom, modulating its pKa (see [16]). (F5) The lone pair of the amine nitrogen of Lys145 can attack on the C4' atom to form a double-bond, and to release the amine of the second product, D-glutamate.

Comments

(B2) Lys145 acts as a general base to deprotonate the alpha-proton of the amino acid substrate, forming a quinonoid intermediate.
(B3) Lys145 acts as a general acid to protonate the C4' atom of the PLP, leading to the formation of a ketimine intermediate.
(C) Schiff-base deforming by hydration, releasing the first product, pyruvate, and PMP.
(C0) Considering the active site of enzyme (PDB;2daa), a water molecule is unlikely to approach from the side of the active site residues, Tyr31 and Lys145.
(C1) Thus, His100* from the adjacent subunit might act as a general base to activate the water molecule at the si-face side of cofactor.
(C2) The activated water molecule makes a nucleophilic attack on the alpha-carbon atom of the substrate (from the si-face side), forming a carbinolamine intermediate.
(C3) The protonated His100* might transfer the proton to Lys145, through a water molecule and either O3' atom of PLP or Tyr31.
(C4) Lys145 may act as a general acid to protonate the N4' atom of the PLP. Tyr31 may modulate the activity of Lys145.
(C5) The lone pair of the hydroxyl oxygen makes a nucleophilic attack on the C4' atom, whereas His100* acts as a general base to deprotonate the hydroxyl group.
(C6) The protonated His100* might transfer the proton to Lys145, through a water molecule and either O3' atom of PLP or Tyr31. Finally, Lys145 act as a general acid to protonate the N4' atom, releasing the first product, pyruvate and the PMP.
(D) Schiff-base forming of PMP with carbonyl group of the second substrate, 2-oxoglutarate.
(D0) The second substrate, 2-oxoglutarate, is bound to the active site, with the carbonyl oxygen hydrogen-bonded by His100*.
(D1) Lys145 acts as a general base to deprotonate the amine group (or the N4' atom) of PMP.
(D2) The deprotonated amine group (or the N4' atom) of PMP makes a nucleophilic attack on the carbonyl carbon of the substrate, whereas His100* acts as a general acid to protonate the carbonyl oxygen, forming a carbinolamine intermediate.
(D3) The protonated Lys145 might transfer the proton to His100*, through a water molecule and either O3' atom of PLP or Tyr31.
(D4) Lys145 acts as a general base to deprotonate the N4' amine group. Tyr31 may modulate the activity of Lys258.
(D5) The lone pair of the N4' nitrogen atom makes a nucleophilic attack on the carbon atom, whereas His100* acts as a general acid to the hydroxyl group of the carbinolamine intermediate, leading to the cleavage between the carbon and the N4' atom, and to the release of a water moleucle. This reaction gives a ketimine intermediate again.
(E) Isomerization (change in the position of double-bond).
(E1) Glu177 modulates and enhances the activity of the PLP cofactor as an electron sink, which facilitates the abstraction of alpha-proton from the C4' atom of the PLP. At the same time, Tyr31 seems to modulate the pKa of Lys145 sidechain, whereas the O3' atom of PLP interacts with the Schiff-base nitrogen atom, modulating the pKa (see [16]).
(E2) Lys145 acts as a general base to deprotonate the C4' atom of the PLP, leading to the formation of a quinonoid intermediate.
(E3) Lys145 acts as a general acid to protonate the alpha-proton of the amino acid substrate, forming an external aldimine. (As a result, Lys145 must be deprotonated, so that it can act as a general base at the next stage.)
(F) Formation of internal aldimine, leading to the elimination of the product, D-glutamate, from PLP.
(F1) Tyr31 might interact with O3' atom of PLP through a water , when the PLP-aldimine is protonated, modulating and keeping the O3' of PLP negatively charged (see [15], [16]).
(F2) The negatively charged O3' atom of PLP modulates the pKa of the internal aldimine with Lys145 (see [16]).
(F3) The deprotonated amine group of Lys145 makes a nucleophilic attack on the C4' carbon of the PLP of the external aldimine, forming a transient geminal diamine intermediate.
(F4) There must be a general acid, which protonates the N4' nitrogen atom of the external aldimine or the geminal diamine. Considering the active-site structure, The adjacent Lys145 nitrogen may play the role as the general acid to protonate the N4' atom, through a water.
This enzyme belongs to the fold-type IV pyridoxal-phosphate-dependent enzymes (see [17]).
Unlike other PLP-dependent aminotransferases (D00101, D00102 in EzCatDB), the catalytic lysine (Lus145) of this enzyme transfer the pro-R hydrogen at the C4' position of pyridoxal phosphate (PLP) on the re face (see [9], [13]). However, the catalytic residues surrounding PLP of this enzyme is very similar to that of aspartate aminotransferase (D00101 in EzCatDB), according to the literature [15].
This enzyme catalyzes transamination, which is composed of the following reactions (see [16]):
(A) Formation of external aldimine (with amine group of D-alanine).
(B) Isomerization (change in the position of double-bond), forming a ketimine intermediate.
(C) Schiff-base deforming by hydration, releasing the first product, pyruvate, and PMP.
(D) Schiff-base forming of PMP with carbonyl group of the second substrate, 2-oxoglutarate, leaging again to a ketimine intermediate.
(E) Isomerization (change in the position of double-bond).
(F) Formation of internal aldimine, leading to the elimination of the product, D-glutamate, from PLP.
These reactions proceed in the following way:
(A) Formation of external aldimine (with amine group of D-alanine).
(A1) Tyr31 might interact with O3' atom of PLP through a water together with the imine nitrogen of Lys145, when the PLP-aldimine is protonated, modulating and keeping the O3' of PLP negatively charged (see [15], [16]).
(A2) The negatively charged O3' atom of PLP modulates the pKa of the internal aldimine with Lys145 (see [16]).
(A3) The deprotonated amine group of D-alanine makes a nucleophilic attack on the C4' carbon of PLP, forming a transient geminal diamine intermediate (see [16]).
(A4) There must be a general base, which deprotonates the amine group of the previously D-alanine substrate, so that the lone pair of the amine group can attack on the C4' atom to form a double-bond, and to release the amine of the catalytic residue, Lys145. The adjacent Lys145 nitrogen may act as a general base to deprotonate the amine group of the incoming substrate, through a water molecule. (see [16])
(A5) The reaction produces the external aldimine with D-alanine.
(B) Isomerization (change in the position of double-bond), forming a ketimine intermediate.
(B1) Glu177 interacts with the N1 atom of PLP, keeping the protonated state of the pyridinium nitrogen of PLP, so that it can modulate and enhance the activity of the PLP cofactor as an electron sink, which facilitates the abstraction of alpha-proton from the D-alanine covalently bound to the PLP (see [16] & [21]). At the same time, Tyr31 seems to modulate the pKa of Lys145 sidechain, whereas the O3' atom of PLP interacts with the Schiff-base nitrogen atom, modulating its pKa (see [16]).
(F5) The lone pair of the amine nitrogen of Lys145 can attack on the C4' atom to form a double-bond, and to release the amine of the second product, D-glutamate.

Created	Updated
2004-03-18	2009-02-26