PDBsum entry 2hgx

Transferase

PDB id: 2hgx

Contents

Protein chain

357 a.a. *

Ligands

PLP ×2

ACY ×3

EPE

Waters ×363

* Residue conservation analysis

PDB id:

2hgx

Name:

Transferase

Title:

Crystal structure of cys315ala mutant of human mitochondrial branched chain aminotransferase

Structure:

Branched-chain-amino-acid aminotransferase, mitochondrial. Chain: a, b. Synonym: bcatm, placental protein 18, pp18, mitochondrial branched chain aminotransferase. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: bcat2, bcatm, eca40. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.208 R-free: 0.231

Authors:

N.H.Yennawar,S.M.Hutson

Key ref:

N.H.Yennawar et al. (2006). Human mitochondrial branched chain aminotransferase isozyme: structural role of the CXXC center in catalysis. J Biol Chem, 281, 39660-39671. PubMed id: 17050531 DOI: 10.1074/jbc.M607552200

Date:

27-Jun-06 Release date: 24-Oct-06

Protein chains

O15382  (BCAT2_HUMAN) -  Branched-chain-amino-acid aminotransferase, mitochondrial from Homo sapiens

Seq: 392 a.a.

Struc: 357 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.6.1.42 - branched-chain-amino-acid transaminase.
• Pathway: Leucine Biosynthesis
• Reaction: L-leucine + 2-oxoglutarate = 4-methyl-2-oxopentanoate + L-glutamate

L-leucine + 2-oxoglutarate (Bound ligand (Het Group name = ACY) matches with 40.00% similarity) = 4-methyl-2-oxopentanoate + L-glutamate

• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = PLP) matches with 93.75% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M607552200

J Biol Chem 281:39660-39671 (2006)

PubMed id: 17050531

Human mitochondrial branched chain aminotransferase isozyme: structural role of the CXXC center in catalysis.

N.H.Yennawar, M.M.Islam, M.Conway, R.Wallin, S.M.Hutson.

ABSTRACT

Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the beta-turn preceding the CXXC motif when compared with wild type protein. The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate alpha-ketoisocaproate in the pyridoxamine 5'-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5'-phosphate, which inhibits catalysis.

Selected figure(s)

Figure 1.
FIGURE 1. C XX C center of oxidized and mutant hBCATm proteins. A, oxidized WT-hBCATm; B, C315A hBCATm; C, C315A/C318A double mutant hBCATm. In the reduced WT protein, the sulfurs of Cys^315 and Cys^318 form a thiol-thiolate hydrogen bond. Upon mutation or oxidation, the thiol-thiolate interaction does not exist. Instead, the Cys^315 and Cys^318 sulfurs form a disulfide bridge.

Figure 4.
FIGURE 4. Active site structures of C315A mutant hBCATm in complex with N-methylleucine (C315A-NML). A and B show the 2F[o] - F[c] difference Fourier electron density maps of the active site in monomer A and monomer B, respectively. C, the superimposed model of the N-methylleucine-bound WT-hBCATm structure and structure of N-methylleucine-bound C315A mutant hBCATm. The carbon skeletons are in khaki in WT-hBCATm and in green in the single mutant C315A-NML structures.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 39660-39671) copyright 2006.

Figures were selected by an automated process.