Gene cloning and characterization of N-carbamyl-l-glutamic acid amidohydrolase involved in ergothioneine utilization in Burkholderia sp. HME13.

Abstract

Burkholderia sp. HME13 utilizes ergothioneine, a strong antioxidant, as the nitrogen source. We have previously shown that ergothionase (ErtA), thiourocanate hydratase (ErtB), 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid desulfhydrase (ErtC), and hydantoin-5-propionic acid amidohydrolase (ErtD) may be involved in ergothioneine utilization in this strain. In this study, we identified the ertE gene in Burkholderia sp. HME13, which encodes a bivalent metal-dependent N-carbamyl-l-glutamic acid amidohydrolase. ErtE showed maximum activity at 60°C and pH 7.0 and was stable at temperatures up to 55°C and pH 6.5-8.0. The Km and Vmax values of ErtE for N-carbamyl-l-glutamic acid were 0.74 mM and 140 U/mg, respectively. EDTA-treated ErtE showed no enzymatic activity, which was restored upon the addition of Co2+, Mn2+, Ni2+, and Fe2+. Expression analyses and enzymatic assays suggested that ErtE is involved in ergothioneine utilization in this strain. Finally, we propose a mechanism for ergothioneine utilization in Burkholderia sp. HME13.