2HDK

Crystal Structure of Cys315Ala-Cys318Ala Mutant of Human Mitochondrial Branched Chain Aminotransferase

PDB DOI: https://doi.org/10.2210/pdb2HDK/pdb

Classification: TRANSFERASE
Organism(s): Homo sapiens
Expression System: Escherichia coli BL21(DE3)
Mutation(s): Yes

Deposited: 2006-06-20 Released: 2006-10-24
Deposition Author(s): Yennawar, N.H., Hutson, S.M.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.40 Å
R-Value Free: 0.237
R-Value Work: 0.182
R-Value Observed: 0.320

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This is version 1.4 of the entry. See complete history.

Literature

Human Mitochondrial Branched Chain Aminotransferase Isozyme: STRUCTURAL ROLE OF THE CXXC CENTER IN CATALYSIS.

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

(2006) J Biol Chem 281: 39660-39671

PubMed: 17050531 Search on PubMed
DOI: https://doi.org/10.1074/jbc.M607552200
Primary Citation of Related Structures:
2HDK, 2HG8, 2HGW, 2HGX, 2HHF

PubMed 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.

Organizational Affiliation

Departments of Biochemistry and Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

Macromolecule Content

Total Structure Weight: 83.29 kDa
Atom Count: 5,979
Modelled Residue Count: 726
Deposited Residue Count: 730
Unique protein chains: 1

Macromolecules

Find similar proteins by:

(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Branched-chain-amino-acid aminotransferase, mitochondrial	A, B	365	Homo sapiens	Mutation(s): 2 EC: 2.6.1.42
UniProt & NIH Common Fund Data Resources
Find proteins for O15382 (Homo sapiens) Explore O15382 Go to UniProtKB: O15382
PHAROS: O15382 GTEx: ENSG00000105552
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	O15382
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 3 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
PLP Query on PLP Download Ideal Coordinates CCD File SDF format, chain C [auth A] SDF format, chain E [auth B] MOL2 format, chain C [auth A] MOL2 format, chain E [auth B]	C [auth A], E [auth B]	PYRIDOXAL-5'-PHOSPHATE C₈ H₁₀ N O₆ P NGVDGCNFYWLIFO-UHFFFAOYSA-N		Interactions Focus chain C [auth A] Focus chain E [auth B] Interactions & Density Focus chain C [auth A] Focus chain E [auth B]
COI Query on COI Download Ideal Coordinates CCD File SDF format, chain F [auth B] MOL2 format, chain F [auth B]	F [auth B]	2-OXO-4-METHYLPENTANOIC ACID C₆ H₁₀ O₃ BKAJNAXTPSGJCU-UHFFFAOYSA-N		Interactions Focus chain F [auth B] Interactions & Density Focus chain F [auth B]
ACY Query on ACY Download Ideal Coordinates CCD File SDF format, chain D [auth A] MOL2 format, chain D [auth A]	D [auth A]	ACETIC ACID C₂ H₄ O₂ QTBSBXVTEAMEQO-UHFFFAOYSA-N		Interactions Focus chain D [auth A] Interactions & Density Focus chain D [auth A]

Binding Affinity Annotations
ID	Source	Binding Affinity
COI	Binding MOAD: 2HDK	Kd: 6.10e+6 (nM) from 1 assay(s)

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 2.40 Å
R-Value Free: 0.237
R-Value Work: 0.182
R-Value Observed: 0.320
Space Group: P 1 2₁ 1

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 59.48	α = 90
b = 112.187	β = 96.16
c = 60.265	γ = 90

Software Package:

Software Name	Purpose
DENZO	data reduction
SCALEPACK	data scaling
CNS	refinement
PDB_EXTRACT	data extraction
AMoRE	phasing

Structure Validation

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Ligand Structure Quality Assessment

Entry History

Deposition Data

Released Date: 2006-10-24

Deposition Author(s):

Yennawar, N.H.

Hutson, S.M.

Revision History (Full details and data files)

Version 1.0: 2006-10-24
Type: Initial release
Version 1.1: 2008-05-01
Changes: Version format compliance
Version 1.2: 2011-07-13
Changes: Non-polymer description, Version format compliance
Version 1.3: 2017-10-18
Changes: Refinement description
Version 1.4: 2021-10-20
Changes: Database references, Derived calculations