PDBsum entry 5tot

Transferase

PDB id: 5tot

Contents

Protein chains

414 a.a.

Waters ×762

PDB id:

5tot

Name:

Transferase

Title:

Crystal structure of aat h143l:h189l double mutant

Structure:

Aspartate aminotransferase, cytoplasmic. Chain: a, b. Synonym: caspat,cysteine aminotransferase,cytoplasmic,cysteine transaminase,ccat,glutamate oxaloacetate transaminase 1,transaminase a. Engineered: yes. Mutation: yes

Source:

Sus scrofa. Pig. Organism_taxid: 9823. Gene: got1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.40Å R-factor: 0.184 R-free: 0.206

Authors:

T.C.Mueser,S.Dajnowicz,A.Kovalevsky

Key ref:

S.Dajnowicz et al. (2017). Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase. J Biol Chem, 292, 5970-5980. PubMed id: 28232482 DOI: 10.1074/jbc.M116.774588

Date:

18-Oct-16 Release date: 01-Mar-17

Protein chains

P00503  (AATC_PIG) -  Aspartate aminotransferase, cytoplasmic from Sus scrofa

Seq: 413 a.a.

Struc: 414 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.6.1.1 - aspartate transaminase.
• Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate
• Cofactor: Pyridoxal 5'-phosphate
• Enzyme class 2: E.C.2.6.1.3 - cysteine transaminase.
• Reaction: L-cysteine + 2-oxoglutarate = 2-oxo-3-sulfanylpropanoate + L-glutamate
• Cofactor: Pyridoxal 5'-phosphate

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

reference

DOI no: 10.1074/jbc.M116.774588

J Biol Chem 292:5970-5980 (2017)

PubMed id: 28232482

Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase.

S.Dajnowicz, J.M.Parks, X.Hu, K.Gesler, A.Y.Kovalevsky, T.C.Mueser.

ABSTRACT

Pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor used to catalyze a wide variety of chemical reactions involved in amino acid metabolism. PLP-dependent enzymes optimize specific chemical reactions by modulating the electronic states of PLP through distinct active site environments. In aspartate aminotransferase (AAT), an extended hydrogen bond network is coupled to the pyridinyl nitrogen of the PLP, influencing the electrophilicity of the cofactor. This network, which involves residues Asp-222, His-143, Thr-139, His-189, and structural waters, is located at the edge of PLP opposite the reactive Schiff base. We demonstrate that this hydrogen bond network directly influences the protonation state of the pyridine nitrogen of PLP, which affects the rates of catalysis. We analyzed perturbations caused by single- and double-mutant variants using steady-state kinetics, high resolution X-ray crystallography, and quantum chemical calculations. Protonation of the pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization in the PLP, which correlates with the enhancement in catalytic rate in AAT. Thus, PLP activation is controlled by the proximity of the pyridinyl nitrogen to the hydrogen bond microenvironment. Quantum chemical calculations indicate that Asp-222, which is directly coupled to the pyridinyl nitrogen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pKa of the pyridine nitrogen but, more significantly, influence the position of the proton that resides between Asp-222 and the pyridinyl nitrogen. These findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.