Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

Abstract

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in numerous essential cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal (PL)-analogues modified at the 2’-position which are taken up by cells and metabolized in situ. These PL-analogues are phosphorylated to functional cofactor surrogates by cellular PL kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH 4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions

“…The type of reaction catalyzed is dependent on the stereochemistry of the external aldimine intermediate and on which of the three α‐carbon groups is aligned perpendicular with the conjugated ring system, as is described in Dunathan’s hypothesis [1]. PLP‐dependent enzymes are estimated to account for ~ 4% of all enzyme activities, and recent chemo‐proteomic strategies have enabled the identification of unannotated proteins as PLP‐dependent [2]. Recently, we reviewed some of the most common reactions that use PLP as a key cofactor in natural products biosynthesis, where the diversity of catalysis is on full display [3].…”

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues

“…[3] This additional group is capable of forming areversible covalent bond with the e-amino group of aspecific lysine residue. [8] Theuse of functional groups,capable of forming areversible covalent bond with lysine residues,c ould be useful for pharmaceutical applications,p rovided that the reactivity of those chemical moieties was not too high, leading to ablockade by human serum albumin in blood. [6] The latter has been investigated as au seful tool for different applications in chemical biology.For instance,Francis and co-workers had previously investigated the use of PLP and orthohydroxy-pyridinecarboxyaldehydes for site-specific modification of N-terminal amino acids.…”

Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage

“…The metabolically active form is pyridoxal 5Ј-phosphate (PLP). PLP is a crucial cofactor for the activity of over 140 enzymes, several of them involved in amino acid metabolism (66,67). Two different routes for the de novo synthesis of PLP exist in organisms: 1-deoxy-D-xylulose 5-phosphate (DOXP)-depend-ent and DOXP-independent (68).…”