Carboxyspermidine Dehydrogenase of Spermidine Biosynthesis from Bacteroides fragilis

Abstract

The human gut microbiome performs a host of metabolic processes which remain largely unstudied, many of which have implications for human health. For example, human cells uptake the microbial polyamine spermidine ( 1), and increased spermidine levels are associated with the progression of pancreatic and colon cancers ( 2, 3). Thus gut microbe spermidine biosynthesis is a potential therapeutic target. Spermidine is produced by the dominant gut microbes via a unique enzymatic pathway ( 4). The first enzyme in this pathway is carboxyspermidine dehydrogenase (CASDH), an NAD(P)H‐dependent saccharopine dehydrogenase expected to catalyze the reductive condensation of either 1,3‐diaminopropane or putrescine with L‐aspartate semialdehyde to form carboxynorspermidine or carboxyspermidine, respectively ( 5). CASDH enzymes involved in spermidine biosynthesis have not been structurally or kinetically characterized from any species. We propose the characterization of CASDH from a dominant gut microbial species using X‐ray crystallography and steady‐state kinetic analysis. The CASDH gene from Bacteroides fragilis was chosen as this species is known to adhere to colon cancer epithelial cells and has been associated with tumor progression. We have successfully expressed BfCASDH in an E. coli BL21 expression cell line and purified it via affinity chromatography. Crystallization trials and the development of kinetic assays are currently underway. X‐ray crystal structures of BfCASDH will reveal the active site structure and the amino acid residues involved in binding and catalysis. In addition, our kinetic assays will confirm the preferred substrate identities and enzymatic activities of the BfCASDH. These data will contribute basic science knowledge necessary for the future explorations of potential inhibitors.Support or Funding InformationKT was supported by NIH BLaST Undergraduate Research Award RL5GM118990. BLaST is supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, & UL1GM118991.