N -Carbamoylputrescine Amidohydrolase of Bacteroides thetaiotaomicron , a Dominant Species of the Human Gut Microbiota.
Hiromi ShimokawaMikiyasu SakanakaYuki FujisawaHirokazu OhtaYuta SugiyamaShin KuriharaPublished in: Biomedicines (2023)
Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N -carbamoylputrescine amidohydrolase (NCPAH) that converts N -carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His) 6 was analyzed for enzymatic activity and found to be capable of converting N -carbamoylputrescine to putrescine, with a Michaelis constant ( K m ) and turnover number ( k cat ) of 730 µM and 0.8 s -1 , respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron .
Keyphrases
- endothelial cells
- high performance liquid chromatography
- escherichia coli
- cell proliferation
- genome wide
- copy number
- induced pluripotent stem cells
- tandem mass spectrometry
- mass spectrometry
- simultaneous determination
- reactive oxygen species
- solid phase extraction
- gene expression
- dna methylation
- nitric oxide
- genome wide identification
- room temperature
- high resolution