Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central 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 analogues modified at the 2′-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 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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Supplementary information, chemical characterization and proteomic data analysis are available in the online version of the paper. Crystallographic data of alanine racemase structures have been deposited in the Protein Data Bank68 (www.rcsb.org) under PDB codes 6G56, 6G58 and 6G59. The proteomic MS data (raw data and MaxQuant output tables for protein groups and peptides) have been deposited in the ProteomeXchange Consortium69 (http://proteomecentral.proteomexchange.org) via the PRIDE70 partner repository (data set identifier: PXD006483).
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This project received funding from the European Research Council (ERC) and the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 725085, CHEMMINE, ERC consolidator grant). Further financial support includes doctoral scholarships to A.H. from the Deutscher Akademischer Austausch Dienst (DAAD) and to M.S. from the Studienstiftung des Deutschen Volkes. The authors thank the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for the supply of the Nebraska Transposon Mutant Library (NTML). The authors also thank M. Wolff and K. Bäuml for technical assistance, the Swiss Light Source (SLS) and European Synchrotron Radiation Facility (ESRF) for beam time, and the staff of beamlines PX I (SLS), ID23-2 and ID29 (ESRF) for setting up of the beamlines for data collection. The authors thank M. H. Wright and B. M. Williams for critical proofreading of the manuscript.
The authors declare no competing interests.
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Hoegl, A., Nodwell, M.B., Kirsch, V.C. et al. Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics. Nature Chem 10, 1234–1245 (2018). https://doi.org/10.1038/s41557-018-0144-2
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