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.

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The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains.