The chaperone role of the pyridoxal 5′-phosphate and its implications for rare diseases involving B6-dependent enzymes

Highlights

• PLP plays a catalytic role in a variety of distinct enzymatic reactions.

• PLP plays a chaperone role during the in vitro folding process of some PLP-enzymes.

• Some inherited diseases due to mutations of PLP-enzymes are treated with pyridoxine.

• The chaperone role of PLP for pathogenic variants of ALAS2, AGT and DDC is described.

Abstract

The biologically active form of the B6 vitamers is pyridoxal 5′-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme–coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed.

Introduction

Vitamers B6 include pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their related 5′-phosphate derivatives (PNP, PMP, PLP). Their structures are reported in Fig. 1. Humans as well as other animals cannot synthesize PLP by a de novo pathway. Nevertheless, vitamin B6 is present in a wide variety of foods, like meat, milk products, potatoes, beans, nuts and several fruits and vegetables [1]. In animal products it is primarily found as PLP and PMP, whereas plant-derived products mostly contain PN(P). Vitamers B6 acquired from diet are recycled in a “salvage pathway” involving the ATP-dependent pyridoxal kinase (PLK), the flavin mononucleotide (FMN)-dependent pyridoxine (pyridoxamine) oxidase (PNPOx), and several different phosphatases (PP) (Fig. 1). In a recent paper, Albersen et al. have demonstrated that the intestine plays a substantial role in human vitamin B6 metabolism [2]: PLK converts PL, PN and PM into the corresponding phosphorylated vitamers, while PNPOx converts PNP and PMP into PLP. Then, PLP is dephosphorylated by an ecto-enzyme intestinal phosphatase, and by portal circulation is delivered to the liver where it is reconverted to PLP by PLK. About 60% of the vitamers in blood is PLP bound to albumin, while the rest is PN, PM, PL. Since to enter the cells and pass the blood–brain barrier the vitamers must be dephosphorylated, PLP is converted again into PL by a membrane associated tissue-nonspecific alkaline phosphatase. On the other hand, PL enters the neurons at choroid plexus, is transported to cerebrospinal fluid through an active mechanism, and crosses the neuron plasma membranes to be re-phosphorylated inside the cells [3].

The biologically active form of vitamin B6 is PLP, which acts as a coenzyme in more than 160 distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. PLP represents one of nature's most versatile cofactors. In the past 70 years extensive researches have been mainly focused on the structure–function relationships of PLP-enzymes [4], [5], [6], [7]. In these enzymes, PLP is invariably bound as an imine to the ε-amino group of a lysyl residue of the protein in a structure known as the internal aldimine. After the initial binding of the substrate amino acid as a Michaelis complex, an external aldimine is formed between the C4′ of the cofactor and the α-amino group of the substrate, which substitutes in the Schiff base linkage the ε-amino group of the PLP-binding lysine. Transimination itself is not a single step process. Indeed, it proceeds through a geminal diamine in which both enzyme and substrate amino groups are bound to C4′. These steps are shared by all the multiple reactions catalysed by PLP-enzymes. Then, depending on the particular enzyme, one of the bonds to the α-carbon of the amino acid is heterolytically cleaved through delocalization of the pair of bonding electrons into the conjugated system of the Schiff base linkage and the pyridine ring of the coenzyme [6], [8]. Dunathan hypothesis [9] predicts that the course of the reaction at this stage would depend on which of the three bonds to the α-carbon is held perpendicular to the plane of the coenzyme-imine π system by interactions between apoenzyme and substrate. Thus, the reaction catalysed depends on which of the three α-substituents is lost, i.e. (i) elimination of CO₂ from Cα (α-decarboxylation), (ii) deprotonation (transamination, racemization, β-elimination and β-replacement, γ-elimination and γ-replacement), and (iii) elimination of the side chain of amino acids (α-synthesis and aldolic cleavage) (Fig. 2). In any case, this event results in the formation of a quinonoid intermediate in which the substrate and the cofactor generate a coplanar structure. Thus, in all PLP-enzymes PLP has two basic chemical properties: (1) is linked through its aldehyde group to a lysine residue at the active site, and (2) acts as an electron sink withdrawing electrons from the substrate. One exception to this common mechanism has been reported for PLP-dependent phosphorylases. In members of this class of enzymes, PLP does not act as an electrophylic catalyst but it participates in proton transfer through its phosphate group [10].