EzCatDB D00105

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

Synonyms

EC 2.6.1.21
D-amino acid aminotransferase
D-amino acid transaminase
DAAT
D-aspartate aminotransferase

Pfam

PF01063 (Aminotran_4)
[Graphical view]

KEGG pathways

MAP code

Pathways

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

UniProtKB:Accession Number

P19938

Entry name

DAAA_BACYM

Activity

D-alanine + 2-oxoglutarate = pyruvate + D- glutamate.

Subunit

Homodimer.

Subcellular location

Cofactor

Pyridoxal phosphate.

Compound table: links to PDB-related databases & PoSSuM

Cofactors

Substrates

Products

intermediates

KEGG-id

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

amino acids,carboxyl group

ChEBI

PubChem

Unbound

Unbound

Unbound

Unbound

Unbound

Analogue:ACT

Unbound

1g2wB01

Unbound

Analogue:ACT

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Unbound

Analogue:PMP

Unbound

Intermediate-bound:PMP

1a0gB02

Analogue:PMP

Unbound

Intermediate-bound:PMP

1daaA02

Bound:PLP

Unbound

1daaB02

Bound:PLP

Unbound

1g2wA02

Bound:PLP

Unbound

1g2wB02

Bound:PLP

Unbound

2daaA02

Analogue:DCS

Unbound

Intermediate-analogue:DCS

Unbound

2daaB02

Analogue:DCS

Unbound

Intermediate-analogue:DCS

Unbound

2dabA02

Bound:PLP

Unbound

2dabB02

Bound:PLP

Unbound

3daaA02

Analogue:PDD

Unbound

Intermediate-bound:PDD

Unbound

3daaB02

Analogue:PDD

Unbound

Intermediate-bound:PDD

Unbound

4daaA02

Bound:PLP

Unbound

4daaB02

Bound:PLP

Unbound

5daaA02

Bound:PLP

Unbound

5daaB02

Bound:PLP

Unbound

Active-site residues

resource

literature [8], [16], [19], [21]

pdb

Catalytic residues

Cofactor-binding residues

comment

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

TYR 31;HIS 100

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

Fig.1

Fig.1, Fig.8, p.4964

Fig.1, p.765

Fig.1, p.617-618

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]

PubMed ID

4950474

Journal

Adv Enzymol Relat Areas Mol Biol

Year

1971

Volume

Pages

79-134

Authors

Dunathan HC

Title

Stereochemical aspects of pyridoxal phosphate catalysis.

[2]

PubMed ID

2713327

Journal

Biochemistry

Year

1989

Volume

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.

[3]

PubMed ID

2496746

Journal

Biochemistry

Year

1989

Volume

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.

[4]

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.

[5]

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.

[6]

PubMed ID

1902115

Journal

Biochemistry

Year

1991

Volume

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.

[7]

PubMed ID

1445909

Journal

Biochemistry

Year

1992

Volume

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.

[8]

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.

[9]

Comments

X-RAY CRYSTALLOGRAPHY (1.94 ANGSTROMS)

Medline ID

95352651

PubMed ID

7626635

Journal

Biochemistry

Year

1995

Volume

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

Related UniProtKB

PubMed ID

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.

[11]

PubMed ID

8580849

Journal

Protein Sci

Year

1995

Volume

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.

[12]

PubMed ID

8652553

Journal

Biochemistry

Year

1996

Volume

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.

[13]

PubMed ID

9063963

Journal

Biosci Biotechnol Biochem

Year

1996

Volume

Pages

181-7

Authors

Yoshimura T, Jhee KH, Soda K

Title

Stereospecificity for the hydrogen transfer and molecular evolution of pyridoxal enzymes.

[14]

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.

[15]

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.

[16]

Comments

X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS)

Medline ID

98206914

PubMed ID

9538014

Journal

Biochemistry

Year

1998

Volume

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

Related UniProtKB

PubMed ID

Journal

Curr Opin Struct Biol

Year

1998

Volume

Pages

759-69

Authors

Jansonius JN

Title

Structure, evolution and action of vitamin B6-dependent enzymes.

[18]

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.

[19]

Comments

X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)

Medline ID

98420361

PubMed ID

9749913

Journal

Protein Eng

Year

1998

Volume

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]

Journal

J Microbiol Biotechnol

Year

1999

Volume

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.

[21]

Comments

X-ray crystallography

PubMed ID

9930994

Journal

Biochemistry

Year

1999

Volume

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

[22]

PubMed ID

10800595

Journal

Adv Enzymol Relat Areas Mol Biol

Year

2000

Volume

Pages

129-84

Authors

Mehta PK, Christen P

Title

The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

[23]

PubMed ID

10630999

Journal

Biochemistry

Year

2000

Volume

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.

[24]

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.

[25]

PubMed ID

10673430

Journal

Structure Fold Des

Year

2000

Volume

Pages

R1-6

Authors

Schneider G, Kack H, Lindqvist Y

Title

The manifold of vitamin B6 dependent enzymes.

[26]

PubMed ID

11933244

Journal

Chem Rec

Year

2001

Volume

Pages

373-84

Authors

Soda K, Yoshimura T, Esaki N

Title

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions.

[27]

PubMed ID

11642362

Journal

Prog Nucleic Acid Res Mol Biol

Year

2001

Volume

Pages

175-206

Authors

Hutson S

Title

Structure and function of branched chain aminotransferases.

[28]

PubMed ID

12297014

Journal

J Biochem Mol Biol

Year

2002

Volume

Pages

306-12

Authors

Ro HS

Title

Effects of salts on the conformation and catalytic properties of d-amino acid aminotransferase.

comments

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]).
(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.
(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

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 - )