PDBsum entry 4cbs

Transferase

PDB id: 4cbs

Contents

Protein chain

385 a.a.

Ligands

PLP

Waters ×82

PDB id:

4cbs

Name:

Transferase

Title:

X-ray structure of quintuple mutant of human alanine glyoxylate aminotransferase, agxt_rheam

Structure:

Serine--pyruvate aminotransferase. Chain: a. Synonym: spt, alanine--glyoxylate aminotransferase, agt, alanine glyoxylate aminotransferase. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 511693. Expression_system_variant: plyss

Resolution:

2.30Å R-factor: 0.223 R-free: 0.257

Authors:

C.Yunta,A.Albert

Key ref:

N.Mesa-Torres et al. (2014). The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase. Biochem J, 462, 453-463. PubMed id: 24957194 DOI: 10.1042/BJ20140250

Date:

16-Oct-13 Release date: 09-Jul-14

Protein chain

P21549  (SPYA_HUMAN) -  Alanine--glyoxylate aminotransferase from Homo sapiens

Seq: 392 a.a.

Struc: 385 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.6.1.44 - alanine--glyoxylate transaminase.
• Reaction: glyoxylate + L-alanine = glycine + pyruvate
• Cofactor: Pyridoxal 5'-phosphate

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

• Enzyme class 2: E.C.2.6.1.51 - serine--pyruvate transaminase.
• Reaction: L-serine + pyruvate = 3-hydroxypyruvate + L-alanine
• Cofactor: Pyridoxal 5'-phosphate

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

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: 10.1042/BJ20140250

Biochem J 462:453-463 (2014)

PubMed id: 24957194

The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

N.Mesa-Torres, C.Yunta, I.Fabelo-Rosa, J.M.Gonzalez-Rubio, J.M.Sánchez-Ruiz, E.Salido, A.Albert, A.L.Pey.

ABSTRACT

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.