title: Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.
abstract: We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.
authors.affiliation: Institute for Research on Earth Evolution (IFREE), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan. takano@jamstec.go.jp
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Takano
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Chikaraishi
authors.forename: Nanako O
authors.initials: NO
authors.lastname: Ogawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitazato
authors.forename: Naohiko
authors.initials: N
authors.lastname: Ohkouchi
mesh_terms: D000409:Alanine / Q000032:analysis / Q000302:isolation & purification*; D001419:Bacteria / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D009587:Nitrogen Isotopes / Q000032:analysis*; D010457:Peptidoglycan / Q000032:analysis*; D013237:Stereoisomerism; D014633:Valine / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D009587:Nitrogen Isotopes; D010457:Peptidoglycan; D014633:Valine; D000409:Alanine