De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1
Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the ‘dark side’ of metabolism.
This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1).
We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR (1H, 13C, 1H-1H-COSY, 1H-13C-HSQC, 1H-13C-HMBC and 1H-15N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation.
We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium.
Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.
KeywordsFunctional genomics Bacterial metabolism LC/MS-Orbitrap NMR Structure elucidation
We thank Jean-François Gallard from Institut de Chimie des Substances Naturelles (CNRS, Gif-sur-Yvette, France) for his expertise in NMR. The authors would like to thank Olek Maciejak and Marie-Jeanne Clément (SABNP, INSERM U 1204—Université d’Evry Val-d’Essonne, Université Paris-Saclay, France) for assistance in 1H and 13C NMR experiments. We are grateful to Cécile Robert–Ansart who was involved as part of a student internship in the project.
MS and AP conceived the study. MT, LS, ED, CL, EP and PLS conducted experiments. MT, LS, ED, CL, EP, JCT, PLS and AP analyzed data. MT, ED, JCT, PLS and AP wrote the manuscript. All authors read and approved the manuscript.
This work was supported by grants from the Commissariat à l’Energie Atomique et aux Energies Alternatives, CNRS, and Université Evry-Val-d’Essonne/Université Paris-Saclay, and by the Region Ile de France for financial support of the 600 MHz NMR spectrometer.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
- Afonso, C., Cole, R. B., & Tabet, J. C. (2010) Dissociation of even-electron ions. In Wiley (Ed.), Electrospray and MALDI mass spectrometry. New York: WileyGoogle Scholar
- D’Argenio, D. A., Segura, A., Coco, W. M., Bunz, P. V., & Ornston, L. N. (1999). The physiological contribution of Acinetobacter PcaK, a transport system that acts upon protocatechuate, can be masked by the overlapping specificity of VanK. Journal of Bacteriology, 181, 3505–3515.PubMedPubMedCentralGoogle Scholar
- Kale, N. S., Haug, K., Conesa, P., Jayseelan, K., Moreno, P., Rocca-Serra, P., … Steinbeck, C. (2016). MetaboLights: An open-access database repository for metabolomics data. Current Protocols in Bioinformatics, 53, 14 13 1–18.Google Scholar
- Martin, G., & Williams, A. (2008) Utilizing long-range 1 H- 15 N 2D NMR spectroscopy in chemical structure elucidation. New York: Wiley.Google Scholar