Publication

Article Metrics

Citations


Online attention

Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase

DOI: 10.1016/j.jbc.2021.100795 DOI Help

Authors: Anna Barile (Consiglio Nazionale delle Ricerche; Sapienza Università di Roma) , Theo Battista (Consiglio Nazionale delle Ricerche; Sapienza Università di Roma) , Annarita Fiorillo (Consiglio Nazionale delle Ricerche; Sapienza Università di Roma) , Martino Luigi Di Salvo (Sapienza Università di Roma) , Francesco Malatesta (Sapienza Università di Roma) , Angela Tramonti (Consiglio Nazionale delle Ricerche; Sapienza Università di Roma) , Andrea Ilari (Consiglio Nazionale delle Ricerche) , Roberto Contestabile (Sapienza Università di Roma)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 4

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 21741

Open Access Open Access

Abstract: Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and it can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24 and Arg215) that form an “arginine-cage” and efficiently trap PLP. The crystal structure of the PNPO-PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer, and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate-PLP ternary complex catalytically incompetent. Site directed mutagenesis studies focused on the “arginine cage” validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R215L variant).

Journal Keywords: pyridoxine 5’-phosphate oxidase; vitamin B6 metabolism; bacterial metabolism; enzyme inhibitor; pyridoxal phosphate; allosteric regulation; crystal structure; asymmetry

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography

Added On: 21/05/2021 20:41

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)