N-Carbamoylputrescine Amidohydrolase of Bacteroides thetaiotaomicron, a Dominant Species of the Human Gut Microbiota

Abstract

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 (Km) and turnover number (kcat) 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.

Insights into the action of the pharmaceutical metformin: Targeted inhibition of the gut microbial enzyme agmatinase

“… 29 N-carbamoylputrescine is then transformed into putrescine either by putrescine carbamoyltransferase or N-carbamoylputrescine amidase with the latter being hydrolytic and the former produces carbamoylphosphate that can be used by carbamate kinase to yield ATP ( Figure 4 ). 30 , 31 To see if agmatine catabolism via AgDI is inhibited by metformin or galegine, the previously characterized AgDI homolog from Enterococcus faecalis was expressed in E. coli and tested in vitro. 31 The purified enzyme was active on agmatine but not inhibited by either 50 mM metformin or 5 mM galegine ( Figure S4 ).…”