4A00

Structure of an engineered aspartate aminotransferase

Summary for 4A00

• Entry DOI: 10.2210/pdb4a00/pdb
• Related: 1AAM 1AAW 1AHE 1AHF 1AHG 1AHX 1AHY 1AIA 1AIB 1AIC 1AMQ 1AMR 1AMS 1ARG 1ARH 1ARI 1ARS 1ART 1ASA 1ASB 1ASC 1ASD 1ASE 1ASF 1ASG 1ASL 1ASM 1ASN 1B4X 1BQA 1BQD 1C9C 1CQ6 1CQ7 1CQ8 1CZC 1CZE 1G4V 1G4X 1G7W 1G7X 1IX6 1IX7 1IX8 1QIR 1QIS 1QIT 1SPA 1TOE 1TOG 1TOI 1TOJ 1TOK 1YOO 2AAT 3AAT 3ZZJ 3ZZK 5EAA
• Descriptor: ASPARTATE AMINOTRANSFERASE, ALANYL-PYRIDOXAL-5'-PHOSPHATE, DI(HYDROXYETHYL)ETHER, ... (5 entities in total)
• Functional Keywords: transferase
• Biological source: ESCHERICHIA COLI
• Cellular location: Cytoplasm: P00509
• Total number of polymer chains: 1
• Total formula weight: 44505.96

Authors

Fernandez, F.J.,deVries, D.,Pena-Soler, E.,Coll, M.,Christen, P.,Gehring, H.,Vega, M.C. (deposition date: 2011-09-06, release date: 2011-12-28, Last modification date: 2023-12-20)

Primary citation

Fernandez, F.J.,De Vries, D.,Pena-Soler, E.,Coll, M.,Christen, P.,Gehring, H.,Vega, M.C.
Structure and Mechanism of a Cysteine Sulfinate Desulfinase Engineered on the Aspartate Aminotransferase Scaffold.
Biocim.Biophys.Acta, 1824:339-, 2011

PubMed Abstract: The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

PubMed: 22138634
DOI: 10.1016/J.BBAPAP.2011.10.016

Experimental method

X-RAY DIFFRACTION (2.34 Å)