Preparation and Application of 13 C-Labeled myo -Inositol to Identify New Catabolic Products in Inositol Metabolism in Lactobacillus casei

• Published in: Biochemistry (Easton) 2020-08, Vol.59 (32), p.2974-2985
• Main Authors: Ramos-Figueroa, Josseline S, Aamudalapalli, Hari Babu, Jagdhane, Rajendra C, Smith, Joseph, Palmer, David R J
• Format: Article
• Language: English
• Subjects: Carbon Isotopes - chemistry; Inositol - chemistry; Inositol - metabolism; Lactobacillus casei - metabolism; Oxidation-Reduction
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.0c00539

-Inositol (mI) is widely distributed in all domains of life and is important for several cellular functions, including bacterial survival. The enzymes responsible for the bacterial catabolism of mI, encoded in the operon, can vary from one organism to another, and these pathways have yet to be fully characterized. We previously identified a new -inositol dehydrogenase (sIDH) in the operon of that can oxidize mI in addition to the natural substrate, -inositol, but the product of mI oxidation was not determined. Here we report the identification of these metabolites by monitoring the reaction with C nuclear magnetic resonance. We prepared all six singly C-labeled mI isotopomers through a biocatalytic approach and used these labeled inositols as substrates for sIDH. The use of all six singly labeled mI isotopomers allowed for metabolite characterization without isolation steps. sIDH oxidation of mI produces 1l-5- -inosose preferentially, but also two minor products, 1d- -inosose and 1l- -inosose. Together with previous crystal structure data for sIDH, we were able to rationalize the observed oxidation preference. Our relatively simple procedure for the preparation of isotopically labeled mI standards can have broad applications for the study of mI biotransformations.