PDBsum entry 1czc

Transferase

PDB id: 1czc

Contents

Protein chain

396 a.a. *

Ligands

PLP

GUA

Waters ×160

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Cocrystallization of a mutant aspartate aminotransferase with a c5-Dicarboxylic substrate analog: structural comparison with the enzyme-C4-Dicarboxylic analog complex.

Authors

S.Oue, A.Okamoto, T.Yano, H.Kagamiyama.

Ref.

J Biochem (tokyo), 2000, 127, 337-343.

PubMed id

10731702

Abstract

A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.

Secondary reference #1

Title

Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-Active site residues.

Authors

S.Oue, A.Okamoto, T.Yano, H.Kagamiyama.

Ref.

J Biol Chem, 1999, 274, 2344-2349. [DOI no: 10.1074/jbc.274.4.2344]

PubMed id

9891001

Figure 2.
Fig. 2. Superimposition of the ATB17-isovalerate complex (pink, large domain; gray, small domain) and the wild-type AspAT-maleate complex (purple). The backbone of one subunit of ATB17, of which the large domain (residues 49-325) was superimposed on that of the wild-type AspAT (31), is indicated by a thick line. The NH[2]- and COOH termini of the subunit are indicated (N and C). The side chains of the residues that were mutated in ATB17 are shown as follows: the two clusters of the residues (see text) are in red and green, and the other residues are in light blue. The coenzyme, pyridoxal 5'-phosphate (yellow), and the bound valine analog, isovalerate (dark blue), are also shown. Maleate of the wild-type AspAT-maleate complex is omitted. Figs. 2 and 4 were produced with MOLSCRIPT and RASTER 3D (32-34).

Figure 4.
Fig. 4. Close-up views of the active site and the two clusters of the mutated residues. The residues are colored as shown in Fig. 2. The structure of the ATB17-isovalerate complex is indicated by thick gray lines and that of the wild-type AspAT-maleate complex is indicated by thin purple lines. A, several water molecules are introduced into the active site of ATB17 (light blue spheres). One water molecule (WAT1) is located at almost the same position as a water molecule observed in the wild-type AspAT (a purple sphere). Arg292, Val293, and Ser297 belong to the other subunit of the dimer (asterisks). B and C, viewed from the same direction as in Fig. 2.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #2

Title

Directed evolution of an aspartate aminotransferase with new substrate specificities.

Authors

T.Yano, S.Oue, H.Kagamiyama.

Ref.

Proc Natl Acad Sci U S A, 1998, 95, 5511-5515. [DOI no: 10.1073/pnas.95.10.5511]

PubMed id

9576913

Figure 1.
Fig. 1. Sequence analysis of the evolved AspATs that have high activity for 2-oxovaline. (A) Lines show the entire coding region of the aspC gene. Longer bars indicate the nucleotide positions of missense mutations, which caused amino acid substitutions, and shorter bars indicate those of silent mutations. Asterisks are the missense mutations conserved in all five mutant AspATs. (B) Amino acid substitutions. Residues were numbered according to the sequence of cytosolic AspAT from pig as described (27).

Figure 3.
Fig. 3. Stereo representation of the structure of the wild-type E. coli AspAT complexed with 2-methyl-L-aspartate (16). The side chains of the six functionally important residues mutated in this study are shown by full bonds (Asn34, Ile^37, Ser139, Asn142, Asn297, and Val387). The coenzyme pyridoxal 5'-phosphate, the substrate analog, and the side chains of Trp140 and Arg386 are also shown (open bonds). Asn297 belongs to the other subunit of the dimer (asterisk).