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

Anna Barile,Theo Battista,Annarita Fiorillo,Martino Luigi Di Salvo,Francesco Malatesta,Angela Tramonti,Andrea Ilari,Roberto Contestabile

doi:10.1016/j.jbc.2021.100795

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 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/R21L variant).

Highlights

Pyridoxal 5’-phosphate (PLP) acts as cofactor for over 150 enzymes (1, 2) involved in a number of metabolic pathways such as the synthesis, transformation, and degradation of amines and amino acids; supply of one carbon units; transsulfuration; synthesis of tetrapyrrolic compounds and polyamines; and biosynthesis and degradation of neurotransmitters
From the environment and protein turnover. The latter route is the only one available in organisms that cannot synthesize PLP, such as humans, and they obtain it through a salvage pathway (Fig. 1) catalyzed by the enzymes pyridoxal kinase, pyridoxine 5’-phosphate oxidase (PNPO; EC 1.4.3.5), and either specific or nonspecific phosphatases (3)
We have recently demonstrated (5) that Escherichia coli PNPO (ePNPO) undergoes an allosteric feedback inhibition by PLP, through a mechanism in which binding of substrate at the active site and binding of PLP at an allosteric site negatively affect each other to a large extent (Fig. 2A). Such a strong allosteric coupling is reflected in the lack of catalytic competence of the enzyme– PNP–PLP ternary complex (PES in Fig. 2A) and in the lack of capability by the enzyme–PLP complex (PE, in which PLP is bound at the allosteric site) to bind a second PLP molecule at the active site

Summary

Introduction

Pyridoxal 5’-phosphate (PLP) acts as cofactor for over 150 enzymes (1, 2) involved in a number of metabolic pathways such as the synthesis, transformation, and degradation of amines and amino acids; supply of one carbon units; transsulfuration; synthesis of tetrapyrrolic compounds (including heme) and polyamines; and biosynthesis and degradation of neurotransmitters. Such values are similar to that obtained with the wildtype enzyme (69 ± 19%), strongly suggesting that the putative secondary PLP-binding site indicated by previous crystallographic studies does not correspond to the allosteric tight binding site.

Results

Conclusion