Structural Determination of a New Peptidolipid Family from Rhodococcus opacus and the Pathogen Rhodococcus equi by Multiple Stage Mass Spectrometry

The cell walls of the genus Rhodococcus including the pathogenic bacterium Rhodococcus equi (R. equi) and biotechnologically important bacterium Rhodococcus opacus (R. opacus) contain an abundant peptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MSⁿ) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MSⁿ(n = 2-8) on the [M + Na]⁺ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in R. opacus is defined as C₁₇H₃₅CH(OH)CH₂CO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a β-OH fatty acyl (C₁₈-C₂₂) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH-CO bond. By contrast, the main peptidolipids found in R. equi belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the β-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the -COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Peypoux, F. et al. Biochemistry 1978, 17, 3992-3996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.